U.S. patent application number 16/978542 was filed with the patent office on 2021-01-21 for treatment of demyelinating diseases.
The applicant listed for this patent is University of Kansas, Victoria Link Ltd.. Invention is credited to Bronwyn Maree Kivell, Anne Camille La Flamme, Thomas Edward Prisinzano.
Application Number | 20210015813 16/978542 |
Document ID | / |
Family ID | 1000005163752 |
Filed Date | 2021-01-21 |
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United States Patent
Application |
20210015813 |
Kind Code |
A1 |
Kivell; Bronwyn Maree ; et
al. |
January 21, 2021 |
TREATMENT OF DEMYELINATING DISEASES
Abstract
The present invention relates generally to methods of using
nalfurafine for treating and/or preventing demyelinating disease in
a subject, and in particular for treating and/or preventing
multiple sclerosis (MS). Also disclosed is nalfurafine for use in
treating and/or preventing MS as well as pharmaceutical
compositions and unit dosage forms comprising nalfurafine for use
for treating and/or preventing demyelinating disease in a subject,
and in particular for treating and/or preventing MS.
Inventors: |
Kivell; Bronwyn Maree;
(Wellington, NZ) ; La Flamme; Anne Camille;
(Wellington, NZ) ; Prisinzano; Thomas Edward;
(Lawrence, KS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Victoria Link Ltd.
University of Kansas |
Kelburn, Wellington
Lawrence |
KS |
NZ
US |
|
|
Family ID: |
1000005163752 |
Appl. No.: |
16/978542 |
Filed: |
March 7, 2019 |
PCT Filed: |
March 7, 2019 |
PCT NO: |
PCT/IB2019/051870 |
371 Date: |
September 4, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/485 20130101;
A61P 25/28 20180101 |
International
Class: |
A61K 31/485 20060101
A61K031/485; A61P 25/28 20060101 A61P025/28 |
Goverment Interests
1. U.S. GOVERNMENT RIGHTS
[0001] This invention was made with government support under
DA018151 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2018 |
AU |
2018900754 |
Claims
1.-26. (canceled)
27. A method of treating a demyelinating disease in a subject,
comprising administering a pharmaceutical composition comprising
nalfurafine to the subject.
28. The method according to claim 27, wherein the pharmaceutical
composition is administered to the subject for a period of (i) at
least 7 days, (ii) at least 14 days, or (iii) at least one
month.
29. The method according to claim 27, wherein the pharmaceutical
composition is administered to the subject to obtain a dosage of
about 0.1 to about 10.0 .mu.g nalfurafine daily.
30. The method according to claim 27, wherein the pharmaceutical
composition is administered to the subject to obtain a dosage of
nalfurafine which is equivalent to a dose of about 0.003 to about
0.3 mg/kg of nalfurafine in mice.
31. The method according to claim 27, wherein the demyelinating
disease is selected from the group consisting of multiple sclerosis
(MS), optic neuritis, Devic's disease, inflammatory demyelinating
diseases, central nervous system neuropathies, myelopathies, Tabes
dorsalis, leukoencephalopathies, leukodystrophies, Guillain-Barre
syndrome and its chronic counterpart, chronic inflammatory
demyelinating polyneuropathy, anti-MAG (myelin-associated
glycoprotein) peripheral neuropathy, Charcot Marie Tooth (CMT)
disease, copper deficiency, progressive inflammatory neuropathy,
and any combination thereof.
32. The method according to claim 31, wherein administration of the
pharmaceutical composition to the subject results in one or more
clinical outcomes selected from the group consisting of: a decrease
or delay in nerve cell demyelination; a healing of damaged nerve
tissue; an increase in remyelination of demyelinated nerves in the
subject's central nervous system; neuroprotection; protection of
damaged nerve tissue from further disease activity; promotion of
neuronal outgrowth in the subject's central nervous system; and an
improvement in nerve function.
33. The method according to claim 31, wherein administration of the
pharmaceutical composition to the subject results in one or more
clinical outcomes selected from the group consisting of: a decrease
in demyelinating disease progression; a decrease in demyelinating
disease severity; a decrease in frequency or severity of relapsing
demyelinating disease attacks; a decrease in disability caused by
demyelinating disease; a decrease in demyelinating disease clinical
symptoms; a decrease in paralysis; an improvement in balance or
coordination; and an enhanced rate of remission.
34. The method according to claim 31, wherein administration of the
pharmaceutical composition to the subject results in a reduction of
one or more clinical symptoms selected from the group consisting
of: loss of sensitivity, muscle weakness, impaired walking,
impaired hand function, pronounced reflexes, muscle spasms,
difficulty in moving, ataxia, spasticity, problems with speech or
swallowing, visual problems, fatigue, acute or chronic pain, facial
pain, incontinence, reduced cognitive ability, depression, anxiety,
sexual dysfunction, Uhthoff s phenomenon, and Lhermitte's sign.
35. A method of increasing remyelination of nerves in a subject,
comprising administering a pharmaceutical composition comprising
nalfurafine to the subject.
36. The method according to claim 35, wherein the pharmaceutical
composition is administered to the subject for a period of (i) at
least 7 days, (ii) at least 14 days, or (iii) at least one
month
37. The method according to claim 35, wherein the pharmaceutical
composition is administered to the subject to obtain a dosage of
about 0.1 to about 10.0 .mu.g nalfurafine daily.
38. The method according to claim 35, wherein the pharmaceutical
composition is administered to the subject to obtain a dosage of
nalfurafine which is equivalent to a dose of about 0.003 to about
0.3 mg/kg of nalfurafine in mice.
39. The method according to claim 35, wherein the subject has one
or more symptoms of a demyelinating disease selected from the group
consisting of multiple sclerosis (MS), optic neuritis, Devic's
disease, inflammatory demyelinating diseases, central nervous
system neuropathies, myelopathies, Tabes dorsalis,
leukoencephalopathies, leukodystrophies, Guillain-Barre syndrome
and its chronic counterpart, chronic inflammatory demyelinating
polyneuropathy, anti-MAG (myelin-associated glycoprotein)
peripheral neuropathy, Charcot Marie Tooth (CMT) disease, copper
deficiency, progressive inflammatory neuropathy, and any
combination thereof.
40. The method according to claim 39, wherein administration of the
pharmaceutical composition to the subject results in one or more
clinical outcomes selected from the group consisting of: a decrease
or delay in nerve cell demyelination; a healing of damaged nerve
tissue; an increase in remyelination of demyelinated nerves in the
subject's central nervous system; neuroprotection; protection of
damaged nerve tissue from further disease activity; promotion of
neuronal outgrowth in the subject's central nervous system; and an
improvement in nerve function.
41. The method according to claim 39, wherein administration of the
pharmaceutical composition to the subject results in one or more
clinical outcomes selected from the group consisting of: a decrease
in demyelinating disease progression; a decrease in demyelinating
disease severity; a decrease in frequency or severity of relapsing
demyelinating disease attacks; a decrease in disability caused by
demyelinating disease; a decrease in demyelinating disease clinical
symptoms; a decrease in paralysis; an improvement in balance or
coordination; and an enhanced rate of remission.
42. The method according to claim 39, wherein administration of the
pharmaceutical composition to the subject results in a reduction of
one or more clinical symptoms selected from the group consisting
of: loss of sensitivity, muscle weakness, impaired walking,
impaired hand function, pronounced reflexes, muscle spasms,
difficulty in moving, ataxia, spasticity, problems with speech or
swallowing, visual problems, fatigue, acute or chronic pain, facial
pain, incontinence, reduced cognitive ability, depression, anxiety,
sexual dysfunction, Uhthoff s phenomenon, and Lhermitte's sign.
43. A method of attenuating demyelination of nerves in a subject,
comprising administering a pharmaceutical composition comprising
nalfurafine to the subject.
44. The method according to claim 43, wherein the pharmaceutical
composition is administered to the subject for a period of (i) at
least 7 days, (ii) at least 14 days, or (iii) at least one
month.
45. The method according to claim 43, wherein the pharmaceutical
composition is administered to the subject to obtain a dosage of
about 0.1 to about 10.0 .mu.g nalfurafine daily.
46. The method according to claim 44, wherein the pharmaceutical
composition is administered to the subject to obtain a dosage of
nalfurafine which is equivalent to a dose of about 0.003 to about
0.3 mg/kg of nalfurafine in mice.
47. The method according to claim 43, wherein the subject has one
or more symptoms of a demyelinating disease selected from the group
consisting of multiple sclerosis (MS), optic neuritis, Devic's
disease, inflammatory demyelinating diseases, central nervous
system neuropathies, myelopathies, Tabes dorsalis,
leukoencephalopathies, leukodystrophies, Guillain-Barre syndrome
and its chronic counterpart, chronic inflammatory demyelinating
polyneuropathy, anti-MAG (myelin-associated glycoprotein)
peripheral neuropathy, Charcot Marie Tooth (CMT) disease, copper
deficiency, progressive inflammatory neuropathy, and any
combination thereof.
48. The method according to claim 46, wherein administration of the
pharmaceutical composition to the subject results in one or more
clinical outcomes selected from the group consisting of: a decrease
or delay in nerve cell demyelination; a healing of damaged nerve
tissue; an increase in remyelination of demyelinated nerves in the
subject's central nervous system; neuroprotection; protection of
damaged nerve tissue from further disease activity; promotion of
neuronal outgrowth in the subject's central nervous system; and an
improvement in nerve function.
49. The method according to claim 46, wherein administration of the
pharmaceutical composition to the subject results in one or more
clinical outcomes selected from the group consisting of: a decrease
in demyelinating disease progression; a decrease in demyelinating
disease severity; a decrease in frequency or severity of relapsing
demyelinating disease attacks; a decrease in disability caused by
demyelinating disease; a decrease in demyelinating disease clinical
symptoms; a decrease in paralysis; an improvement in balance or
coordination; and an enhanced rate of remission.
50. The method according to claim 46, wherein administration of the
pharmaceutical composition to the subject results in a reduction of
one or more clinical symptoms selected from the group consisting
of: loss of sensitivity, muscle weakness, impaired walking,
impaired hand function, pronounced reflexes, muscle spasms,
difficulty in moving, ataxia, spasticity, problems with speech or
swallowing, visual problems, fatigue, acute or chronic pain, facial
pain, incontinence, reduced cognitive ability, depression, anxiety,
sexual dysfunction, Uhthoff s phenomenon, and Lhermitte's sign.
Description
2. TECHNICAL FIELD
[0002] The disclosure relates generally to the use of nalfurafine
(NaIF) in the prevention and treatment of demyelinating diseases,
in particular, multiple sclerosis.
3. BACKGROUND
[0003] The myelin sheath covers important nerve fibres in the
central and peripheral nervous system of mammals, helping to
facilitate transmission of neural impulses. Diseases that affect
myelin interrupt these nerve transmissions. The developing myelin
sheath can be affected by congenital metabolic disorders such as
phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Hurler's
syndrome, and Krabbe's disease. Demyelination can also occur in
adults as a result of injury, metabolic disorders, immune attack,
ischemia and toxic agents.
[0004] Demyelination impairs conduction of signals to the affected
nerves, causing deficiency of sensation, movement, cognition and
other functions. Demyelination of the central nervous system is
associated with multiple sclerosis (MS), Devic's disease, acute
disseminated encephalomyelitis, adrenoleukodystrophy,
leukoencephalopathy and Leber's optiv atrophy. Demyelination of the
peripheral nervous symptom gives rise to diseases such as
Guillain-Barre syndrome, chronic inflammatory demyelinating
polyneuropathy, Charcot Marie Tooth (CMT) disease and progressing
inflammatory neuropathy.
[0005] Multiple sclerosis (MS) is the most well-known demyelination
disease, affecting about 2.5 million people worldwide. Sufferers
endure a range of symptoms including fatigue, vision problems,
numbness, cognitive impairment, incontinence, poor balance and
muscle weakness, ultimately leading to paralysis. MS can follow
four major disease courses, each of which can be mild, moderate or
severe: [0006] 1. Relapsing-Remitting MS (RRMS)--clearly defined
attacks (flare-ups) of worsening neurological function followed by
partial or complete remission [0007] 2. Primary-Progressive MS
(PPMS)--slowly worsening neurological function at variable rates,
with no distinct remission [0008] 3. Secondary-Progressing MS
(SPMS)--an initial period of RRMS is followed by a steady
worsening, with or without flare-ups and remissions [0009] 4.
Progressive-Relapsing MS (PRMS)--steadily worsening neurological
function with clear flare-ups and partial or no remission.
[0010] While there is no cure for MS, many FDA approved drugs such
as beta-interferon and glatiramer acetate are used to reduce
relapse rates and the formation of new lesions. Unfortunately,
current treatments are not very successful in preventing the
disability associated with MS and are more successful in treating
RRMS than other types. For example, current drugs are unable to
stop or reverse disease progression and disability. Clearly,
alternative treatments for MS are needed.
[0011] It is therefore an object of the present invention to go at
least some way towards meeting this need in the art, to provide
products and methods useful in the treatment of the disability
associated with MS and/or that are able to stop and/or reverse MS
disease progression and disability and/or to at least to provide
the public with a useful choice.
4. SUMMARY OF THE INVENTION
[0012] In one aspect the invention provides a pharmaceutical
composition comprising nalfurafine and pharmaceutically acceptable
excipients for treating a demyelinating disease in a subject in
need thereof.
[0013] In one aspect the invention provides a pharmaceutical
composition comprising nalfurafine and at least one
pharmaceutically acceptable excipient for use for treating a
demyelinating disease in a subject in need thereof.
[0014] In another aspect the invention provides unit dosage forms
comprising about 0.01 to about 5 mg of nalfurafine and at least one
pharmaceutically acceptable carrier or excipient. In one
embodiment, the unit dosage form comprises 0.05 to about 2.0 mg of
nalfurafine and at least one pharmaceutically acceptable carrier or
excipient. In one embodiment the unit dosage form comprises about
0.15 to about 0.6 mg nalfurafine and at least one pharmaceutically
acceptable carrier or excipient.
[0015] In another aspect the invention provides a method of
treating a demyelinating disease in a subject in need thereof,
comprising administering a therapeutically effective amount of
nalfurafine to the subject.
[0016] In another aspect the invention provides a method of
treating a demyelinating disease in a subject comprising
identifying a subject who would benefit from a decreased level of
demyelination and administering to the subject a therapeutically
effective amount of an agent that decreases the level of
demyelination in the subject relative to the level of demyelination
before administering the agent, wherein the agent comprises
nalfurafine.
[0017] In another aspect the invention provides a method of
treating a demyelinating disease in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of an agent that decreases the level of demyelination in the
subject relative to the level of demyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0018] In another aspect the invention provides a method of
increasing remyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject.
[0019] In another aspect the invention provides a method of
increasing remyelination in a subject comprising identifying a
subject who would benefit from an increased level of remyelination
and administering to the subject a therapeutically effective amount
of an agent that increases the level of remyelination in the
subject relative to the level of remyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0020] In another aspect the invention provides a method of
increasing remyelination in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
[0021] The invention also provides a use of nalfurafine in the
manufacture of a medicament for treating a demyelinating disease in
a subject in need thereof.
[0022] The invention also provides a use of nalfurafine in the
manufacture of a medicament for increasing remyelination in a
subject in need thereof.
[0023] The invention also provides nalfurafine for use for treating
a demyelinating disease.
[0024] The invention also provides nalfurafine for use for
increasing remyelination.
[0025] In one embodiment the disease is a demyelinating
myelinoclastic disease.
[0026] In one embodiment the disease is a demyelinating
leukodystrophic disease.
[0027] In one embodiment the demyelinating disease is a central
nervous system demyelinating disease. In one embodiment the central
nervous system demyelinating disease is selected from the group
comprising MS (including clinically isolated syndrome; CIS), optic
neuritis, Devic's disease, inflammatory demyelinating diseases,
central nervous system neuropathies, myelopathies like Tabes
dorsalis, leukoencephalopathies, leukodystrophies, or a combination
thereof.
[0028] In one embodiment the demyelinating disease is MS.
[0029] In another embodiment the demyelinating disease is a
peripheral nervous system demyelinating disease. In one embodiment
the peripheral nervous system demyelinating disease is elected from
the group comprising Guillain-Barre syndrome and its chronic
counterpart, chronic inflammatory demyelinating polyneuropathy,
anti-myelin associated glycoprotein (MAG) peripheral neuropathy,
Charcot Marie Tooth (CMT) disease, copper deficiency and
progressive inflammatory neuropathy.
[0030] In another aspect the invention provides a method of
attenuating demyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject and thereby attenuating a level of demyelination in the
subject relative to the level of demyelination when nalfurafine is
not administered.
[0031] The invention also provides a use of nalfurafine in the
manufacture of a medicament for attenuating demyelination in a
subject in need thereof. In one embodiment, the subject is a human
with MS.
[0032] The invention also provides nalfurafine for use for
attenuating demyelination in a subject in need thereof.
[0033] In another aspect the invention provides a method of
treating MS in a subject in need thereof, comprising administering
a therapeutically effective amount of nalfurafine to the
subject.
[0034] In another aspect the invention provides a method of
treating MS in a subject in need thereof, comprising administering
to the subject a therapeutically effective amount of an agent that
decreases a level of demyelination in the subject relative to the
level before administering the agent and/or that increases a level
of remyelination in the subject in the subject relative to the
level before administering the agent, wherein the agent comprises
nalfurafine.
[0035] The invention also provides a use of nalfurafine in the
manufacture of a medicament for treating MS in a subject in need
thereof.
[0036] The invention also provides nalfurafine for use for treating
MS in a subject in need thereof.
[0037] In one embodiment the subject has RRMS. In one embodiment
the subject has PPMS. In one embodiment the subject has, or is
diagnosed as having, SPMS. In one embodiment the subject has, or is
diagnosed as having, PRMS. In one embodiment the subject has, or is
diagnosed as having, Clinically Isolated Syndrome (CIS).
[0038] In one embodiment the treatment of MS results in one or more
clinical outcomes when compared to subjects not treated with
nalfurafine selected from the group consisting of: [0039] (a) a
decrease in MS disease progression; [0040] (b) a decrease in MS
disease severity; [0041] (c) a decrease in nerve cell
demyelination; [0042] (d) a decrease in frequency or severity of
relapsing MS attacks; [0043] (e) a decrease in MS clinical
symptoms; [0044] (f) the healing of damaged nerve tissue
(neuro-restoration); [0045] (g) an increase in remyelination of
demyelinated nerves in the central nervous system
(neuro-restoration/protection); [0046] (h) the protection of
damaged nerve tissue from further disease activity
(neuro-protection); [0047] (i) the promotion of neuronal outgrowth
(neuro-regeneration) in the central nervous system; [0048] (j) a
decrease in disability caused by MS; [0049] (k) an improvement of
nerve function; and [0050] (l) an enhanced rate of remission.
[0051] In another embodiment the treatment of MS results in a
reduction of one or more clinical symptoms of MS including, but not
limited to loss of sensitivity or changes in sensation such as
tingling, pins and needles or numbness, muscle weakness or
paralysis of variable severity, very pronounced reflexes, muscle
spasms, or difficulty in moving; difficulties with coordination and
balance (ataxia); spasticity; problems with speech or swallowing,
visual problems (nystagmus, optic neuritis or double vision),
fatigue, acute or chronic pain, neuropathic pain, facial pain
(trigeminal neuralgia), bladder and bowel difficulties,
incontinence, reduced cognitive ability, depression, anxiety and
other emotional abnormalities, sexual dysfunction, Uhthoff's
phenomenon (a worsening of symptoms due to exposure to higher than
usual temperatures), and Lhermitte's sign (an electrical sensation
that runs down the back when bending the neck).
[0052] In one aspect the invention provides a method of
accelerating remission from MS in a subject in need thereof, the
method comprising administering a therapeutically effective amount
of nalfurafine to the subject.
[0053] In one aspect the invention provides a method of
accelerating remission from MS in a subject in need thereof, the
method comprising administering a therapeutically effective amount
of an agent that decreases the level of demyelination in the
subject relative to the level of demyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0054] In one aspect the invention provides a method of
accelerating remission from MS in a subject in need thereof, the
method comprising administering a therapeutically effective amount
of an agent that increases the level of remyelination in the
subject relative to the level of remyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0055] The invention also provides a use of nalfurafine in the
manufacture of a medicament for accelerating remission from MS in a
subject in need thereof.
[0056] The invention also provides nalfurafine for use for
accelerating remission from MS in a subject in need thereof.
[0057] In another aspect the invention provides a method of
treating a demyelinating disease in a subject comprising
identifying a subject who would benefit from a decreased level of
demyelination and administering to the subject a therapeutically
effective amount of an agent that decreases the level of
demyelination relative to the level of demyelination before
administering the agent, wherein the agent comprises
nalfurafine.
[0058] In another aspect the invention provides a method of
increasing remyelination in a subject comprising identifying a
subject who would benefit from an increased level of remyelination
and administering to the subject a therapeutically effective amount
of an agent that increases the level of remyelination relative to
the level of remyelination before administering the agent, wherein
the agent comprises nalfurafine.
[0059] In the above methods of the invention:
[0060] In one embodiment the therapeutically effective amount for a
subject is equivalent to a dose of about 0.003 to about 0.3
mg/kg/day in mice.
[0061] In one embodiment the subject is human. In one embodiment
the method comprises administering about 0.01 to about 5 .mu.g
nalfurafine daily, about 0.01 to about 4 .mu.g, about 0.01 to about
3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to about 2
.mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1 .mu.g,
about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5 .mu.g, or
about 0.25 .mu.g nalfurafine daily.
[0062] In some embodiments the method comprises administering less
than about 1 .mu.g nalfurafine, preferably less than 1 ug
nalfurafine daily.
[0063] In some embodiments the method comprises a long duration
therapy.
[0064] In some embodiments the long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 5 days, at least 6 days,
or at least 7 days.
[0065] In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least a week, at least 2 weeks,
at least 3 weeks, at least 4 weeks, at least 6 weeks, or at least 8
weeks.
[0066] In some embodiments the long duration therapy comprises
administration for at least 5 days, at least 6 days, at least 7
days, at least 14 days, at least 21 days, at least 28 days, at
least 35 days, at least 42 days, at least 45 days, at least 60
days, at least 120 days, at least 240 days, or at least 360
days.
[0067] In some embodiments the long duration therapy comprises a
dosing gap of at least 1 day.
[0068] Other aspects of the invention may become apparent from the
following description which is given by way of example only and
with reference to the accompanying figures.
[0069] In this specification where reference has been made to
patent specifications, other external documents, or other sources
of information, this is generally for the purpose of providing a
context for discussing the features of the invention. Unless
specifically stated otherwise, reference to such external documents
is not to be construed as an admission that such documents, or such
sources of information, in any jurisdiction, are prior art, or form
part of the common general knowledge in the art. However, these
external documents and references are all cited herein by reference
in their entireties or at least to the extent described herein.
[0070] It is intended that reference to a range of numbers
disclosed herein (for example, 1 to 10) also incorporates reference
to all rational numbers within that range (for example, 1, 1.1, 2,
3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of
rational numbers within that range (for example, 2 to 8, 1.5 to 5.5
and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges
expressly disclosed herein are hereby expressly disclosed. These
are only examples of what is specifically intended and all possible
combinations of numerical values between the lowest value and the
highest value enumerated are to be considered to be expressly
stated in this application in a similar manner.
[0071] Whenever a range is given in the specification, for example,
a temperature range, a time range, or a composition range, all
intermediate ranges and subranges, as well as all individual values
included in the ranges given are intended to be included in the
disclosure. In the disclosure and the claims, "and/or" means
additionally or alternatively. Moreover, any use of a term in the
singular also encompasses plural forms.
5. BRIEF DESCRIPTION OF THE DRAWINGS
[0072] The invention will now be described by way of example only
and with reference to the drawings in which:
[0073] FIG. 1 is a graph showing the progression of disease in mice
which have experimental autoimmune encephalomyelitis (EAE) over 45
days, wherein the mice in Example 1 were treated with 0.01, 0.03,
0.1 or 0.3 mg/kg nalfurafine daily from onset (day 17).
[0074] FIG. 2 is two graphs showing the total disability of EAE
mice over (A) 45 days and (B) 18 days wherein the mice in Example 2
were treated with 0.03, 0.1 or 0.3 mg/kg nalfurafine daily from
onset (day 17).
[0075] FIG. 3 is a graph showing the % weight change of EAE mice in
Example 3 over 45 days wherein the mice were treated with 0.03, 0.1
or 0.3 mg/kg nalfurafine daily from onset (day 17).
[0076] FIG. 4 is three graphs showing immune cell infiltration into
the brain of EAE mice in Example 4 after 45 days, wherein the mice
were treated with 0.03, 0.1 or 0.3 mg/kg nalfurafine daily from
onset (day 17).
[0077] FIG. 5 is a graph showing the progression of disease in EAE
mice in Example 5 over 45 days, wherein the mice, which had not yet
developed EAE, were treated with 0.03, 0.1 or 0.3 mg/kg nalfurafine
daily from onset (day 17).
[0078] FIG. 6 is a series of Transmission Electron Microscope (TEM)
images of spinal cord sections from EAE mice in Example 6 after 45
days, wherein the mice were treated with 0.03 mg/kg nalfurafine
daily from onset (day 17).
[0079] FIG. 7 is a graph showing weight gain over 65 days of mice
in Example 7 treated with 0.3% cuprizone for 5 weeks, wherein the
mice were treated with 0.1 mg/kg nalfurafine daily from week 4.
[0080] FIG. 8 is a graph showing the rotarod performance score of
mice in Example 8 at 9 weeks treated with cuprizone for 5 weeks,
wherein the mice were treated with 0.1 mg/kg nalfurafine daily from
week 4.
[0081] FIG. 9 is a series of TEM imagines of the corpus callosum of
mice in Example 9 at 9 weeks treated with cuprizone for 5 weeks,
wherein the mice were treated with 0.1 mg/kg nalfurafine daily from
week 4.
[0082] FIG. 10 shows that nalfurafine promotes functional recovery
from paralysis when administered therapeutically (at disease onset)
in the experimental autoimmune encephalomyelitis (EAE) model of
MS.
[0083] FIG. 11 shows that nalfurafine is not effective when
administered therapeutically as a short 4-day course starting at
disease onset in EAE model of MS.
[0084] FIG. 12 shows that nalfurafine does not alter peak disease
when administered therapeutically in the EAE model of MS.
[0085] FIG. 13 shows that nalfurafine promotes full recovery from
EAE-induced paralysis when administered therapeutically.
[0086] FIG. 14 shows that nalfurafine promotes full recovery from
EAE-induced paralysis when administered therapeutically with an
EC50 for % recovery of <0.001 mg/kg.
[0087] FIG. 15 shows that nalfurafine promotes sustained recovery
from EAE-induced paralysis when administered therapeutically.
[0088] FIG. 16 shows that nalfurafine also promotes functional
recovery from paralysis in male mice when administered
therapeutically in EAE model of MS.
[0089] FIG. 17 shows that nalfurafine also promotes full recovery
in male mice when administered therapeutically in EAE model of
MS.
[0090] FIG. 18 shows that nalfurafine promotes sustained recovery
in male mice from EAE-induced paralysis when administered
therapeutically.
[0091] FIG. 19 shows that nalfurafine reduces the immune cell
infiltration into the brain when administered therapeutically in
the EAE model of MS (A) whereas U 50488 does not (B).
[0092] FIG. 20 shows that myelination is improved in mice treated
with nalfurafine after the onset of paralysis in the EAE model of
MS.
[0093] FIG. 21 shows that nalfurafine does not alter the proportion
of major lymphocyte populations in the spleen during the chronic
phase of EAE.
[0094] FIG. 22 shows that nalfurafine does not alter the overall
number of CD4 T helper cells in the spleen but shifts the CD4 T
cells from an effector to memory phenotype being suggestive of
immune resolution during the chronic phase of EAE.
[0095] FIG. 23 shows that nalfurafine reduces disease but does not
enable full recovery when the kappa opioid receptor (KOR) is
blocked.
[0096] FIG. 24 shows that activation of the KOR is required for
full recovery from paralysis mediated by nalfurafine.
[0097] FIG. 25 shows that myelination is improved in mice treated
with nalfurafine after the onset of paralysis in the EAE model of
MS.
[0098] FIG. 26 shows that nalfurafine treatment decreases cellular
infiltration into the spinal cord when administered therapeutically
in the EAE model of MS.
[0099] FIG. 27 shows that nalfurafine treatment reduces the level
of activated astrocytes in the spinal cord when administered
therapeutically in the EAE model of MS.
[0100] FIG. 28 shows nalfurafine treatment enhances recovery from
weight loss when administered therapeutically in the cuprizone
model of MS.
[0101] FIG. 29 shows that nalfurafine treatment enhances
remyelination in the brain when administered after demyelination in
the cuprizone demyelination disease model of MS.
[0102] FIG. 30 shows nalfurafine is more effective at promoting
functional recovery than clemastine fumarate, a known remyelinating
drug.
[0103] FIG. 31 shows that nalfurafine promotes a greater and more
sustained recovery than clemastine fumarate, a known remyelinating
drug.
[0104] FIG. 32 shows that nalfurafine promotes recovery in pain
threshold when administered after demyelination in the cuprizone
demyelination disease model of MS.
6. DETAILED DESCRIPTION
6.1 Nalfurafine
[0105] Nalfurafine is a drug commonly prescribed for treatment of
uremic pruritus in people with chronic kidney disease. It is a
non-narcotic opioid with selective K-opioid receptor (KOR) agonist
activity. The inventors have now found that nalfurafine is a
surprisingly effective treatment for demyelinating diseases.
[0106] The generic name "nalfurafine" refers to the compound:
##STR00001##
[0107] The IUPAC name for nalfurafine is
(E)-N-[(4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-dihydroxy-1,2,4,5,-
6,7,7a,13-octahydro-4,12-methanobenzofuro[3,2-e]isoquinoline-7-yl]-3-(fura-
n-3-yl)-N-methylprop-2-enamide. Its CAS number is 152657-84-6.
Nalfurafine HCl may also be referred to as
17-cyclopropylmethyl-3,14-beta-dihydroxy-4,5-alpha-epoxy-6beta-(N-methyl--
trans-3-(3-furyl)acrylamido)morphinan hydrochloride, TRK 820,
AC-820 and MT-9938.
[0108] As used herein the term "nalfurafine" refers to the compound
identified above as well as to its pharmaceutically acceptable
salts and solvates.
[0109] The term "pharmaceutically acceptable salts" refers to salts
prepared from pharmaceutically acceptable non-toxic bases or acids
including inorganic or organic bases and inorganic or organic
acids. Salts derived from inorganic bases include aluminum,
ammonium, calcium, copper, ferric, ferrous, lithium, magnesium,
manganic salts, manganous, potassium, sodium, zinc, and the like.
Particularly preferred are the ammonium, calcium, magnesium,
potassium, and sodium salts. Salts in the solid form may exist in
more than one crystal structure and may also be in the form of
hydrates. Salts derived from pharmaceutically acceptable organic
non-toxic bases include salts of primary, secondary, and tertiary
amines, substituted amines including naturally occurring
substituted amines, cyclic amines, and basic ion exchange resins,
such as arginine, betaine, caffeine, choline,
N,N'-dibenzylethylene-diamine, diethylamine, 2-diethylaminoethanol,
2-dimethylamino-ethanol, ethanolamine, ethylenediamine,
N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,
histidine, hydrabamine, isopropylamine, lysine, methylglucamine,
morpholine, piperazine, piperidine, polyamine resins, procaine,
purines, theobromine, triethylamine, trimethylamine,
tripropylamine, tromethamine, and the like. When nalfurafine is
basic, salts can be prepared from pharmaceutically acceptable
non-toxic acids, including inorganic and organic acids. Such acids
include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric,
ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic,
hydrochloric, isethionic, lactic, maleic, malic, mandelic,
ethanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric,
succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like.
Particularly preferred are citric, hydrobromic, hydrochloric,
maleic, phosphoric, sulfuric, fumaric, and tartaric acids.
[0110] The term "solvate" refers to an aggregate that consists of a
solute ion or molecule with one or more solvent molecules.
"Solvates" include hydrates, that is, aggregates of a compound of
interest with water.
[0111] Nalfurafine can be purchased from small molecule suppliers
such as Med Chem Express, Monmouth Junction and New Jersey, USA;
AdooQ BioScience, Irvine Calif., USA.
6.2 Pharmaceutical Compositions of Nalfurafine
[0112] There is a lack of effective treatments for demyelinating
diseases, including MS, and in particular, there are few effective
agents that act to reduce demyelination and/or to increase
remyelination. Surprisingly, the inventors have found that
pharmaceutical compositions containing nalfurafine can be used to
treat demyelination diseases including but not limited to MS by
acting to increase remyelination and/or to decrease
demyelination.
[0113] Accordingly, in one aspect the invention provides a
pharmaceutical composition comprising nalfurafine and
pharmaceutically acceptable excipients for treating a demyelinating
disease in a subject in need thereof.
[0114] In another aspect the invention provides a pharmaceutical
composition comprising nalfurafine and at least one
pharmaceutically acceptable excipient for use for treating a
demyelinating disease in a subject in need thereof.
[0115] This term "pharmaceutical composition" as used herein
encompasses a product comprising one or more active agents, and
pharmaceutically acceptable excipients comprising inert
ingredients, as well as any product which results, directly or
indirectly, from combination, complexation or aggregation of any
two or more of the ingredients, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. In general,
pharmaceutical compositions are prepared by bringing the active
agent into association with a liquid carrier, a finely divided
solid carrier or both, and then, if necessary, shaping the product
into the desired formulation. Said compositions are prepared
according to conventional mixing, granulating, or coating methods,
respectively, and contain a percentage (%) of the active ingredient
and can be determined by a skilled worker in view of the art.
[0116] The term "comprising" as used herein means "consisting at
least in part of". When interpreting each statement in this
specification that includes the term "comprising", features other
than that or those prefaced by the term may also be present.
Related terms such as "comprise" and "comprises" are to be
interpreted in the same manner.
[0117] The term "consisting essentially of" as used herein means
the specified materials or steps and those that do not materially
affect the basic and novel characteristic(s) of the claimed
invention.
[0118] The term "consisting of" as used herein means the specified
materials or steps of the claimed invention, excluding any element,
step, or ingredient not specified in the claim.
[0119] By "pharmaceutically acceptable excipient" or
"pharmaceutically acceptable carrier" it is meant that the
excipient or carrier must be compatible with the other ingredients
of the formulation and not harmful to the subject to whom the
composition is administered.
[0120] Pharmaceutical compositions as described herein can be
administered topically, orally or parenterally.
[0121] For example, the pharmaceutical compositions can be
administered orally, including sublingually, in the form of
capsules, tablets, elixirs, solutions, suspensions, or boluses
formulated to dissolve in, for example, the colon or duodenum. The
formulations can comprise excipients such as starch or lactose or
flavouring, preserving or colouring agents.
[0122] The pharmaceutical compositions can be injected
parenterally, for example, intravenously, intramuscularly or
subcutaneously. For parenteral administration, the compositions can
be formulated in a sterile aqueous solution or suspension that
optionally comprises other substances, such as salt or glucose.
[0123] The compositions can be administered topically, in the form
of sterile creams, gels, pour-on or spot-on formulations,
suspensions, lotions, ointments, dusting powders, drug-incorporated
dressings, shampoos, collars or transdermal patches. For example,
the compositions as described herein can be incorporated into a
cream comprising an aqueous or oily emulsion of polyethylene
glycols or liquid paraffin; an ointment comprising a white wax soft
paraffin base; a hydrogel with cellulose or polyacrylate
derivatives or other suitable viscosity modifiers; a dry powder;
aerosol with butane, propane, HFA, or CFC propellants; a dressing,
such as, a tulle dressing, with white soft paraffin or polyethylene
glycol impregnated gauze dressings or with hydrogel, hydrocolloid,
or alginate film dressings. The compositions can also be
administered intra-ocularly as an eye drop with appropriate
buffers, viscosity modifiers (for example, cellulose derivatives),
and preservatives (for example, benzalkonium chloride).
[0124] The pharmaceutical compositions as described herein can also
be incorporated into a transdermal patch comprising nalfurafine.
Details of such patches can be found in, for example,
WO2015/025766, WO2015/025767, WO2016/208729, WO2017/094337 and
WO2017/170933, the details of which are incorporated by reference
herein.
[0125] For oral administration, capsules, boluses, or tablets can
be prepared by mixing the pharmaceutical compositions as described
herein with a suitable finely divided diluent or carrier,
additionally containing a disintegrating agent and/or binder such
as starch, lactose, talc, or magnesium stearate.
[0126] For parenteral administration injectable formulations can be
prepared in the form of a sterile solution or emulsion.
[0127] The compositions described herein can be presented in unit
dosage form and can be prepared by any of the methods well known in
the art of pharmacy. The term "unit dosage form" means a single
dose wherein all active and inactive ingredients are combined in a
suitable system, such that the patient or person administering the
drug can open a single container or package with the entire dose
contained therein and does not have to mix any components together
from two or more containers or packages. Typical examples of unit
dosage forms are tablets or capsules for oral administration or
transdermal patches comprising the unit dosage. These examples of
unit dosage forms are not intended to be limiting in any way, but
merely to represent typical examples in the pharmacy arts of unit
dosage forms.
[0128] In another aspect the invention provides unit dosage forms
comprising about 0.01 to about 5 mg of nalfurafine and at least one
pharmaceutically acceptable carrier or excipient. In one
embodiment, the unit dosage form comprises 0.05 to about 2.0 mg of
nalfurafine and at least one pharmaceutically acceptable carrier or
excipient. In one embodiment the unit dosage form comprises about
0.15 to about 0.6 mg nalfurafine and at least one pharmaceutically
acceptable carrier or excipient.
[0129] In one aspect the invention provides a unit dosage form
comprising about 0.1 to about 10 .mu.g of nalfurafine and at least
one pharmaceutically acceptable carrier or excipient. In one
embodiment the unit dosage form comprises about 0.5 to about 7.5
.mu.g nalfurafine, about 0.75 to about 5 .mu.g nalfurafine, about 1
to 4 .mu.g nalfurafine, about 2-3 .mu.g nalfurafine, about 2 .mu.g
nalfurafine, about 3 .mu.g nalfurafine, about 4 .mu.g nalfurafine
or about 5 .mu.g nalfurafine.
[0130] In one embodiment the unit dosage form comprises less than
about 2 .mu.g, 1.5 .mu.g, 1.0 .mu.g, 0.5 .mu.g, 0.25 .mu.g or 0.1
.mu.g, preferably less than 2 .mu.g, 1.5 .mu.g, 1.0 .mu.g, 0.5
.mu.g, 0.25 .mu.g or 0.1 .mu.g.
[0131] In another embodiment, the unit dosage form is for treating
a demyelinating disease in a subject in need thereof, preferably
wherein the subject has MS. In another embodiment, the unit dosage
is formulated for treating a demyelinating disease in a subject in
need thereof. In one embodiment the demyelinating disease is
MS.
[0132] In another embodiment the unit dose is formulated for
increasing remyelination in a subject in need thereof, preferably
wherein the subject has MS.
[0133] In one embodiment, the unit dosage form is for oral
administration, preferably the unit dosage form is formulated for
oral administration. In another embodiment, the unit dosage form is
a transdermal patch.
[0134] The term "about" as used herein means a reasonable amount of
deviation of the modified term such that the end result is not
significantly changed. For example, when applied to a value, the
term should be construed as including a deviation of +/-5% of the
value.
[0135] Pharmaceutical compositions of nalfurafine can be used in
combination with other therapies for treating demyelination
diseases.
6.3 Therapeutic Uses of Nalfurafine
[0136] The inventors have surprisingly found that nalfurafine gives
rise to many positive effects in demyelination in MS mouse models.
For example, the inventors have found that nalfurafine is effective
at treating demyelination in mouse models of EAE and
cuprizone-induced demyelination, results that are translatable to
treating demyelinating diseases such as MS in humans. The inventors
have also found that nalfurafine is unexpectedly effective at
increasing remyelination in subjects in need thereof. Accordingly,
this drug, which has a proven safety record, could be highly
beneficial in the treatment of demyelination diseases and/or for
increasing remyelination.
[0137] As set out in Examples 1, 10 and 12-18, nalfurafine promotes
functional (including full and sustained) recovery from EAE-induced
paralysis in male and female mice. Nalfurafine also reduces
EAE-induced total disability (see Example 2) and promotes recovery
from EAE-induced weight loss (see Example 3). Importantly, the
disease score is reduced completely in the examples described
herein to <0.5, which is considered to represent a "full
recovery" from paralysis in the art, with one exception. A short
4-day time course starting at disease outset was not effective at
promoting recovery (Example 11), demonstrating the efficacy of a
long duration therapy as described herein.
[0138] Nalfurafine reduces immune cell infiltration into the brain
in the EAE model of MS (see Example 4) and is more effective than
the comparator U-50488, which does not (Example 19. When
administered before onset, nalfurafine promotes functional recovery
from paralysis, in the EAE model of MS (see Example 5). Myelination
is also improved in mice treated with nalfurafine after the onset
of paralysis in the EAE model of MS (Examples 6, 20 and 25).
[0139] By the examples described herein the inventors show clearly
that nalfurafine induces and/or increases remyelination in the EAE
model. In Example 6, TEM images of the spinal cords of EAE mice
treated with nalfurafine resemble those of the healthy control.
[0140] The EAE results were confirmed by cuprizone studies
described in Examples 7-9 and 11. In Examples 7 and 28, nalfurafine
improved weight gain when administered after cuprizone-induced
demyelination. In Example 8, nalfurafine enhanced the functional
recovery of coordination and balance in demyelinated mice.
Remyelination of the corpus callosum occurred when
cuprizone-treated mice were administered nalfurafine (see Examples
9 and 29).
[0141] In Example 15 the demonstration of sustained recovery is
noteworthy and shows the quite unexpected ability of nalfurafine to
reverse, in a sustained manner, the symptoms of demyelination. This
surprising result indicates that nalfurafine can mediate sustained
recovery of demyelinating diseases including MS.
[0142] In Example 21, nalfurafine does not deplete the major immune
cell populations in the periphery despite reducing immune cell
infiltration into the brain. In example 22, nalfurafine promotes a
switch in T helper cells from effector to memory cells suggestive
of immune response resolution.
[0143] In Examples 23 and 24, the KOR is required for the full
effect of nalfurafine but nalfurafine is effective at reducing
disease independently of the KOR suggesting the full mechanism by
which nalfurafine exerts its effects is more complex than KOR
activation.
[0144] The positive effects of nalfurafine on mice were
particularly surprising at dosages of 0.003 mg/kg to 0.3 mg/kg,
which can be converted to an equivalent human dose using the
Regan-Shaw equation (Reagan-Shaw S; Nihal M; Ahmad N: Dose
translation from animal to human studies revisited, FASEB J. 2007,
Oct. 17).
[0145] Alternatively, dosages of 0.003 to 0.3 mg/kg can be
converted to an equivalent human dose using the method of
interspecies comparison described herein.
[0146] The skilled worker in the art appreciates that there are
alternative algorithms that can be used to convert an observed
therapeutic dosage from a mouse model into an equivalent human dose
once the effective mouse dosage has been demonstrated. Such
algorithms can be used effectively by the skilled person to
determine the appropriate human dose
[0147] For example, using a method of interspecies comparison, a
skilled worker employs the ratio of the efficacy dose for itch vs
the efficacy dose for MS in the same species. This ratio can be
applied to the human dose to convert dosage for itch to the dosage
for MS. In this case, there is dose data for treating itch in both
mouse and human models, and this enables the calculations described
below.
[0148] Data describing the drug dose that produces 50% of the
maximal effect (ED.sub.50).
TABLE-US-00001 Route of Complete Mouse Adminis- ED.sub.50
Inhibition Model tration (.mu.g/kg) (.mu.g/kg) Reference Substance
P IV 3.77 7.5-10 Winfuran - induced Assessment scratch report
European Substance P SC 1.65 10 Medicines Agency, induced Committee
for scratch Medicinal Products Substance P PO 9.61 100 for Human
induced (66%) Use (EMA/CHMP/ scratch 138212/2014) Morphine SC 2.34
5-10 induced scratch Histamine PO 7.3 30-100 Togashi et al. (2002).
induced Antipruritic activity scratch of the .kappa.-opioid
Substance P PO 19.6 100 receptor agonist, induced TRK-820. Eur J
scratch Pharmacol 435: 259
[0149] For itch model the average in vivo efficacy ED.sub.50 is
.about.2.71 .mu.g/kg (rounded up to 3 .mu.g/kg) by SC or IV
administration (only the data in the top two rows of the table
above were used in this calculation). The rationale for this is:
[0150] Administration in our EAE study was intraperitoneal (i.p.)
[0151] Bioavailability of nalfurafine (as described in
Winfuran--Assessment report European Medicines Agency, Committee
for Medicinal Products for Human Use (EMA/CHMP/138212/2014): [0152]
oral (PO) administration is .about.32% [0153] subcutaneous (s.c.)
is 96% [0154] intravenous (IV) is 100% [0155] Therefore, s.c. and
IV administration will have a similar bioavailability to i.p.,
whereas PO administration will not due to first-pass effect of
hepatic metabolism, and therefore it has been excluded from the
calculations. Additionally, the morphine induced scratch model
works through a mechanism of action unrelated to that of substance
P itch, and therefore was excluded.
Converting Dosage for Itch vs EAE
[0156] The calculation assumes that itch response is a biomarker
(surrogate) for EAE. [0157] 1. Mouse dose for itch is 3
.mu.g/kg/day [0158] 2. Mouse dose for EAE is 3 .mu.g/kg/day
(effective dose shown in FIG. 10) [0159] 3. Therefore, the ratio of
itch to EAE in mouse=1 [0160] 4. Using the ratio of efficacy for
itch vs EAE in the same species (mouse) of 1 [0161] 5. The
effective dose in humans for itch of 2.5 .mu.g/body/day [0162] 6.
Calculation to convert EAE mouse to Human dose prediction:
[0162] EAE mouse dose/(3 .mu.g/kg/day mouse itch.times.2.5
.mu.g/body/day human itch)=Human MS dose. [0163] 7. Conversion of
EAE mouse dose to predicted human MS dose: [0164] 1. 3 .mu.g/kg/day
mouse=2.5 .mu.g/body/day for human (FIG. 10) [0165] 2. 10
.mu.g/kg/day mouse=8.33 .mu.g/body/day for human (FIG. 1) [0166] 3.
30 .mu.g/kg/day mouse=25 .mu.g/body/day for human (FIG. 1) [0167]
4. 100 .mu.g/kg/day mouse=83.33 .mu.g/body/day for human (FIG. 1)
[0168] 5. 300 .mu.g/kg/day mouse=250 .mu.g/body/day for human (FIG.
1)
[0169] As many demyelinating diseases cause horribly debilitating
symptoms, any improvement in treatment outcomes provides an
important development. The inventors have discovered that
nalfurafine is an effective treatment for demyelinating diseases,
and in particular MS. In one example, the inventors believe that
treatment with nalfurafine will be effective for alleviating the
debilitating symptoms related to Clinically Isolated Syndrome
(CIS). One of the MS disease courses, CIS generally refers to a
first episode of neurologic symptoms associated with MS. Typically,
this initial episode is caused by inflammation or demyelination in
the central nervous system (CNS), and will last 24 hours or
more.
[0170] Therefore, in one aspect, the invention provides a method of
treating a demyelinating disease in a subject in need thereof,
comprising administering a therapeutically effective amount of
nalfurafine to the subject.
[0171] In another aspect the invention provides a method of
treating a demyelinating disease in a subject comprising
identifying a subject who would benefit from a decreased level of
demyelination and administering to the subject a therapeutically
effective amount of an agent that decreases the level of
demyelination in the subject relative to the level of demyelination
before administering the agent, wherein the agent comprises
nalfurafine.
[0172] In another aspect the invention provides a method of
treating a demyelinating disease in a subject in need thereof,
comprising administering to the subject a therapeutically effective
amount of an agent that decreases the level of demyelination in the
subject relative to the level of demyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0173] The term "treating" as used herein with reference to a
disease or condition refers to the following: (a) ameliorating the
disease or condition such as by eliminating or causing regression
of or decreasing the severity of the disease or medical condition
of the subject being treated relative to an untreated subject
according to art-accepted criteria for monitoring the disease or
condition (Wattjes et al. (2015). Evidence-based guidelines:
MAGNIMS consensus guidelines on the use of MRI in multiple
sclerosis--establishing disease prognosis and monitoring patients.
Nat. Rev. Neurol. 11, 597-606; Traboulsee et al. (2016). Revised
Recommendations of the Consortium of MS Centers Task Force for a
Standardized MRI Protocol and Clinical Guidelines for the Diagnosis
and Follow-Up of Multiple Sclerosis. AJNR Am. J. Neuroradiol. 37,
394-401; Toosy et al. (2014). Optic neuritis. Lancet Neurol. 13,
83-99; Ontaneda et al. (2017). Clinical outcome measures for
progressive MS trials. Mult. Scler. 23, 1627-1635; Naismith et al.
(2012). Diffusion tensor imaging in acute optic neuropathies:
predictor of clinical outcomes. Arch. Neurol. 69, 65-71); (b)
suppressing the disease or condition such as by slowing or
arresting the development of the disease or condition relative to
an untreated subject according to art-accepted criteria for
monitoring the disease or condition (Oh et al. (2019). Imaging
outcome measures of neuroprotection and repair in MS: A consensus
statement from NAIMS. Neurology; Sormani et al. (2017). Assessing
Repair in Multiple Sclerosis: Outcomes for Phase II Clinical
Trials. Neurother. J. Am. Soc. Exp. Neurother. 14, 924-933; Zhang
et al. (2018). Clinical trials in multiple sclerosis: milestones.
Ther. Adv. Neurol. Disord. 11; Bjartmar et al. (2003). Axonal loss
in the pathology of MS: consequences for understanding the
progressive phase of the disease. J. Neurol. Sci. 206, 165-171;
Toosy et al. (2014). Optic neuritis. Lancet Neurol. 13, 83-99) or
(c) alleviating a symptom of the disease or condition in the
subject relative to an untreated subject according to art-accepted
criteria for monitoring the disease or condition (van Munster et
al. (2017). Outcome Measures in Clinical Trials for Multiple
Sclerosis. CNS Drugs 31, 217-236; Uitdehaag (2018). Disability
Outcome Measures in Phase III Clinical Trials in Multiple
Sclerosis. CNS Drugs 32, 543-558; Toosy et al. (2014). Optic
neuritis. Lancet Neurol. 13, 83-99). In some preferred embodiments
"treating" refers to ameliorating as in (a), suppressing as in (b)
and/or alleviating as in (c) in a statistically significant manner
relative to an appropriate untreated control subject according to
art-accepted criteria for monitoring the disease or condition.
[0174] In the definition of "treating" the art accepted criteria
are one or more of Criteria for measuring disability may include
the expanded disability scale, multiple sclerosis functional
composite Z-score and multiple sclerosis Impact Scale and Medical
Outcomes Study Short Form, imaging of the brain, spinal cord or
optic nerve, Multiple Sclerosis Functional Composite, and novel
composite measures of disability, in addition to tests evaluating
manual dexterity, ambulation, vision (including measures of axial
diffusivity, visual acuity, contrast sensitivity, visual evoked
potentials (VEPs), and thickness of the retinal nerve fiber layer
(RNFL) and cognition.
[0175] The subject may show an observable or measurable decrease in
one or more of the symptoms associated with or related to the
disease or condition as known to those skilled in the art, as
indicating improvement. In some embodiments, the disease or
condition is a demyelinating disease, preferably MS, and the
subject shows an observable and measurable decrease in one or more
of the symptoms associated with or related to MS, preferably a
decrease in demyelination as known to those skilled in the art, as
indicating improvement. In preferred embodiments the improvement is
a statistically significant improvement relative to an appropriate
untreated control subject according to art-accepted criteria for
monitoring the disease or condition.
[0176] The terms "decrease" and "reduced" (and grammatical
variations thereof) as used herein with reference to demyelination
mean any measurable or observable reduction in an amount or level
of demyelination or of any symptom of a demyelinating disease that
is attributable to demyelination in a treated subject relative to
the level of demyelination in an appropriate control (e.g.,
untreated) subject. In preferred embodiments the measurable or
detectable decrease or reduction is a statistically significant
decrease or reduction, relative to an appropriate control.
[0177] The term "increase" (and grammatical variations thereof as
used herein with reference to demyelination means any measurable or
observable increase in an amount or level of remyelination or an
improvement of any symptom of a demyelinating disease that is
attributable to remyelination in a treated subject relative to the
level of remyelination in an appropriate control (e.g., untreated)
subject; e.g., placebo or non-active agent. An example of
quantifying remyelination is demonstrated with treatment with
clemastine fumarate using measures of VEPs to evaluate
remyelination and recovery. (Green et al. (2017) Clemastine
fumarate as a remyelinating therapy for multiple sclerosis
(ReBUILD): a randomised, controlled, double-blind, crossover trial.
Lancet. 390, 2481-2489; Jankowska-Lech et al. (2019). Peripapillary
retinal nerve fiber layer thickness measured by optical coherence
tomography in different clinical subtypes of multiple sclerosis.
Mult. Scler. Relat. Disord. 27, 260-268; Naismith et al. (2012).
Diffusion tensor imaging in acute optic neuropathies: predictor of
clinical outcomes. Arch. Neurol. 69, 65-71; Oh et al. (2019).
Imaging outcome measures of neuroprotection and repair in MS: A
consensus statement from NAIMS. Neurology; Sormani et al. (2017).
Assessing Repair in Multiple Sclerosis: Outcomes for Phase II
Clinical Trials. Neurother. J. Am. Soc. Exp. Neurother. 14,
924-933. In preferred embodiments the measurable or detectable
reduction is a statistically significant reduction, relative to an
appropriate control.
[0178] The terms "administration of" or "administering" should be
understood to mean providing nalfurafine or a pharmaceutical
composition comprising, consisting essentially of, or consisting
of, nalfurafine to the subject in need of treatment in a
therapeutically useful form for the mode of administration.
Nalfurafine can be administered via any suitable route. Potential
routes of administration include without limitation oral,
parenteral (including intramuscular, subcutaneous, intradermal,
intravenous, intraarterial, intramedullary and intrathecal),
intraperitoneal, and topical (including dernnal/epicutaneous,
transdermal, mucosal, transmucosal, intranasal (e.g., by nasal
spray or drop), intraocular (e.g., by eye drop), pulmonary (e.g.,
by inhalation), buccal, sublingual, rectal and vaginal.
[0179] The term "therapeutically" as used herein means "at disease
onset".
[0180] In certain embodiments, nalfurafine is administered via oral
dosage forms such as tablets, capsules, syrups, suspensions, and
the like. In another embodiment, nalfurafine is administered via a
transdermal patch.
[0181] The term "therapeutically effective amount" refers to a
sufficient quantity of the active agent, in a suitable composition,
and in a suitable dosage form to treat the noted disease conditions
or to obtain a measurable or observable result such as a decrease
in demyelination or an increase in remyelination. The
"therapeutically effective amount" will vary depending on the
compound, the severity of the demyelination disease, and the
species, age, weight, etc., of the subject to be treated.
[0182] In one embodiment, the therapeutically effective amount of
nalfurafine is the amount equivalent to about 0.003-about 0.3 mg/kg
in a mouse which can be converted according to accepted practice
into an animal or human subject dosage. For example, using the
Reagan-Shaw equation, a therapeutically effective amount of
nalfurafine for a dog would be about 0.67-about 2 mg/kg.
[0183] In one embodiment, the therapeutically effective amount of
nalfurafine is the amount equivalent to about 0.003-about 0.3 mg/kg
in a mouse, converted according the method of interspecies
comparison described herein. In one embodiment a therapeutically
effective amount of nalfurafine for a human is about 0.01 to about
5 .mu.g nalfurafine daily, preferably about 0.01 to about 2.5 .mu.g
nalfurafine daily.
[0184] In one embodiment the subject is human. In one embodiment
the method comprises administering about 0.01 to about 5 .mu.g
nalfurafine daily, about 0.01 to about 4 .mu.g, about 0.01 to about
3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to about 2
.mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1 .mu.g,
about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5 .mu.g, or
about 0.25 .mu.g nalfurafine daily.
[0185] In one embodiment the method comprises administering about
0.01 to about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2
.mu.g, about 0.05 to about 1 .mu.g, about 0.075 to about 0.75
.mu.g, about 0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325
.mu.g nalfurafine daily.
[0186] In some embodiments the method comprises administering less
than about 1 .mu.g nalfurafine daily, preferably less than 1 .mu.g
nalfurafine daily.
[0187] In one embodiment the method comprises administering about
0.01 to about 0.1 .mu.g nalfurafine daily, about 0.025 to about
0.075 .mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
[0188] In one embodiment the method comprises a long duration
therapy.
[0189] In some embodiments the long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 5 days, at least 6 days,
or at least 7 days.
[0190] In some embodiments the long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 5, preferably at least 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,
preferably at least 90 days.
[0191] In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least a week, at least 2 weeks,
at least 3 weeks, at least 4 weeks, at least 6 weeks, or at least 8
weeks.
[0192] In some embodiments the long duration therapy comprises
administration for at least 5 days, at least 6 days, at least 7
days, at least 14 days, for at least 21 days, for at least 28 days,
for at least 35 days, for at least 42 days, for at least 45 days,
for at least 60 days, for at least 120 days, for at least 240 days,
or for at least 360 days.
[0193] In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 1 week, at least 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, or at least 52
weeks.
[0194] In some embodiments a long duration therapy comprises
administration of a therapeutically effective dose of nalfurafine
to a subject in need thereof for at least 1 month, at least 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or at least
36 months.
[0195] In some embodiments the long duration therapy comprises a
dosing gap, preferably wherein the dosing gap is at least 1
day.
[0196] In some embodiments dosing gap comprises at least 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.
[0197] In some embodiments the dosing gap comprises at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.
[0198] In some embodiments the dosing gap comprises at least 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, or 11 months.
[0199] The term "demyelinating disease" refers to a disease of the
nervous system in which the myelin sheath of neurons is damaged.
Demyelinating diseases include demyelinating myelinoclastic
diseases and demyelinating leukodystrophic diseases. Treatment of a
demyelinating disease can comprise treatment with an agent that
decreases demyelination and/or an agent that increases
remyelination.
[0200] Demyelinating diseases may affect the central nervous system
and peripheral nervous system. The central nervous system
demyelinating diseases include multiple sclerosis including
clinically isolated syndrome (CIS) optic neuritis, Devic's disease,
inflammatory demyelinating diseases, central nervous system
neuropathies like those produced by Vitamin B12 deficiency,
myelopathies like Tabes dorsalis, leukoencephalopathies like
progressive multifocal leukoencephalopathy, leukodystrophies, or a
combination thereof. The peripheral nervous system demyelinating
diseases include Guillain-Barre syndrome and its chronic
counterpart, chronic inflammatory demyelinating polyneuropathy,
anti-MAG peripheral neuropathy, Charcot Marie Tooth (CMT) disease,
copper deficiency, progressive inflammatory neuropathy, or a
combination thereof. The term "subject" refers to a mammal, more
preferably a human, or companion animal. Preferred companion
animals include cats, dogs and horses. Other mammalian subjects
include agricultural animals, including horses, pigs, sheep, goats,
cows, deer, or fowl: and laboratory animal, including monkeys,
rats, mice, rabbits and guinea pig.
[0201] The invention also provides a use of nalfurafine in the
manufacture of a medicament for treating a demyelinating disease in
a subject in need thereof.
[0202] The invention also provides a use of nalfurafine in the
manufacture of a medicament for increasing remyelination in a
subject in need thereof.
[0203] The invention also provides nalfurafine for use for treating
a demyelinating disease.
[0204] The invention also provides nalfurafine for use for
increasing remyelination.
[0205] In one embodiment the disease is a demyelinating
myelinoclastic disease.
[0206] In one embodiment the disease is a demyelinating
leukodystrophic disease.
[0207] In one embodiment the demyelinating disease is a central
nervous system demyelinating disease. In one embodiment the central
nervous system demyelinating disease is selected from the group
comprising MS (including clinically isolated syndrome; CIS), optic
neuritis,
[0208] Devic's disease, inflammatory demyelinating diseases,
central nervous system neuropathies, myelopathies like Tabes
dorsalis, leukoencephalopathies, leukodystrophies, or a combination
thereof.
[0209] In one embodiment the demyelinating disease is MS.
[0210] In another embodiment the demyelinating disease is a
peripheral nervous system demyelinating disease. In one embodiment
the peripheral nervous system demyelinating disease is elected from
the group comprising Guillain-Barre syndrome and its chronic
counterpart, chronic inflammatory demyelinating polyneuropathy,
anti-myelin associated glycoprotein (MAG) peripheral neuropathy,
Charcot Marie Tooth (CMT) disease, copper deficiency and
progressive inflammatory neuropathy.
[0211] In another aspect the invention provides a method of
increasing remyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject.
[0212] In another aspect the invention provides a method of
increasing remyelination in a subject comprising identifying a
subject who would benefit from an increased level of remyelination
and administering to the subject a therapeutically effective amount
of an agent that increases the level of remyelination in the
subject relative to the level of remyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0213] In another aspect the invention provides a method of
increasing remyelination in a subject in need thereof, comprising
administering to the subject a therapeutically effective amount of
an agent that increases the level of remyelination in the subject
relative to the level of remyelination before administering the
agent, wherein the agent comprises nalfurafine.
[0214] Specifically contemplated as embodiments of the invention
described herein relating to a method of increasing remyelination
in a subject are all of the embodiments of the invention set forth
herein relating to the aspects of the invention that are methods of
decreasing demyelination, methods of treating MS, methods of
attenuating demyelination, methods of accelerating remission of MS,
and methods of treating a demyelinating disease.
[0215] In another aspect the invention provides a method of
attenuating demyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of nalfurafine to
the subject and thereby attenuating a level of demyelination in the
subject relative to the level of demyelination when nalfurafine is
not administered.
[0216] In another aspect the invention provides a method of
attenuating demyelination in a subject in need thereof, comprising
administering a therapeutically effective amount of an agent that
decreases the level of demyelination in the subject relative to the
level of demyelination before administering the agent and/or that
increases the level of remyelination in the subject relative to the
level of remyelination before administering the agent wherein the
agent comprises nalfurafine.
[0217] In one embodiment the subject is human. In one embodiment
the method comprises administering about 0.01 to about 5 .mu.g
nalfurafine daily, about 0.01 to about 4 .mu.g, about 0.01 to about
3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to about 2
.mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1 .mu.g,
about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5 .mu.g, or
about 0.25 .mu.g nalfurafine daily.
[0218] In one embodiment the method comprises administering about
0.01 to about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2
.mu.g, about 0.05 to about 1 .mu.g, about 0.075 to about 0.75
.mu.g, about 0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325
.mu.g nalfurafine daily.
[0219] In some embodiments the method comprises administering less
than about 1 .mu.g nalfurafine daily, preferably less than 1 .mu.g
nalfurafine daily.
[0220] In one embodiment the method comprises administering about
0.01 to about 0.1 .mu.g nalfurafine daily, about 0.025 to about
0.075 .mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
[0221] The term "attenuation of demyelination" means in certain
embodiments that the amount or level of demyelination in the
subject as a result of the disease or as a symptom of the disease
is reduced when compared to otherwise identical conditions in an
appropriate control subject or at an appropriate control reference
timepoint and/or in certain embodiments that the amount or level of
remyelination in the subject is increased when compared to an
otherwise identical conditions in an appropriate control subject or
at an appropriate control reference timepoint. In some preferred
embodiments the reduction or increase as compared to the
appropriate control is a statistically significant reduction or
increase.
[0222] In certain preferred embodiments, the term "attenuation of
demyelination" thus means that the amount of or level demyelination
in the subject as a result of the disease or as a symptom of the
disease is reduced or decreased in a statistically significant
manner when compared to a suitable control as would be understood
by a person of skill in the art in view of the present disclosure
and/or the amount or level of remyelination in the subject is
increased in a statistically significant manner when compared to a
suitable control as would be understood by a person of skill in the
art in view of the present disclosure.
[0223] Similarly, the term "improvement in nerve function" refers
to a quantifiable improvement in function having a statistically
different change in a measurable parameter relative to an
appropriate control as recognized by a person of skill in the art.
In some embodiments the improvement in function has a statistically
significant change in the measurable parameter. In one embodiment
the measurable parameter is the disease score as described in
Example 1.
[0224] Symptoms attributable to demyelination will vary depending
on the disease but may include, for example but not limited to,
neurological deficits, such as chronic pain, cognitive impairment
(including memory, attention, conceptualization and problem-solving
skills) and information processing; paresthesia in one or more
extremities, in the trunk, or on one side of the face; weakness or
clumsiness of a leg or hand; or visual disturbances, e.g. partial
blindness and pain in one eye (retrobulbar optic neuritis), dimness
of vision, or scotomas.
[0225] The invention also provides a use of nalfurafine in the
manufacture of a medicament for attenuating demyelination in a
subject in need thereof.
[0226] The invention also provides nalfurafine for use for
attenuating demyelination in a subject in need thereof.
[0227] In another aspect the invention provides a method of
treating MS in a subject in need thereof, comprising administering
a therapeutically effective amount of nalfurafine to the subject.
The subject can suffer from any type of MS including CIS, RRMS,
PRMS, SPMS, PRMS or MS that follows a different and/or undefined
disease course.
[0228] The invention also provides a use of nalfurafine in the
manufacture of a medicament for treating MS in a subject in need
thereof.
[0229] The invention also provides nalfurafine for use for treating
MS in a subject in need thereof.
[0230] In one embodiment the subject has RRMS. In one embodiment
the subject has PPMS. In one embodiment the subject has, or is
diagnosed as having, SPMS. In one embodiment the subject has, or is
diagnosed as having, PRMS. In one embodiment the subject has, or is
diagnosed as having, Clinically Isolated Syndrome (CIS).
[0231] In another aspect the invention provides a method of
treating MS in a subject in need thereof, comprising administering
to the subject a therapeutically effective amount of an agent that
decreases a level of demyelination in the subject relative to the
level before administering the agent and/or that increases a level
of remyelination in the subject in the subject relative to the
level before administering the agent, wherein the agent comprises
nalfurafine.
[0232] In some embodiments the methods of treating MS set forth
herein can comprise one or more of the following steps selected
from the group consisting of diagnosing MS in the subject, testing
for demyelination in the subject, testing for a reduction or
reversal in demyelination in the subject, testing for remyelination
in the subject, testing for a level of paralysis or a reduction or
reversal of a level of paralysis in the subject, and testing for a
decrease or increase of coordination and/or balance in the
subject.
[0233] In one embodiment a method of treating MS and/or of treating
a demyelinating disease and/or of attenuating demyelination and/or
of increasing remyelination comprises identifying a subject who
would benefit from a level of decreased demyelination.
[0234] In some embodiments a subject who would benefit from a level
of decreased demyelination and/or a level of increased
remyelination is identified on the basis of exhibiting one or more
clinical symptoms of MS including, but not limited to: loss of
sensitivity or changes in sensation such as tingling, pins and
needles or numbness, muscle weakness of variable severity, very
pronounced reflexes, muscle spasms, or difficulty in moving;
difficulties with coordination and balance (ataxia); spasticity;
problems with speech or swallowing, visual problems (nystagmus,
optic neuritis or double vision), fatigue, acute or chronic pain,
facial pain (trigeminal neuralgia), bladder and bowel difficulties,
incontinence, reduced cognitive ability, depression, anxiety and
other emotional abnormalities, sexual dysfunction, Uhthoff's
phenomenon (a worsening of symptoms due to exposure to higher than
usual temperatures), and Lhermitte's sign (an electrical sensation
that runs down the back when bending the neck).
[0235] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 5 mg nalfurafine daily, about 0.01 to about 4 .mu.g, about
0.01 to about 3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to
about 2 .mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1
.mu.g, about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5
.mu.g, or about 0.25 .mu.g nalfurafine daily.
[0236] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
[0237] In some embodiments the method comprises administering less
than about 1 .mu.g nalfurafine daily, preferably less than 1 .mu.g
nalfurafine daily.
[0238] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
[0239] In one embodiment the treatment results in one or more
clinical outcomes as compared to subjects not treated with
nalfurafine, selected from the group consisting of: [0240] (a) a
decrease in MS disease progression; [0241] (b) a decrease in MS
disease severity; [0242] (c) a decrease in nerve cell
demyelination; [0243] (d) a decrease in frequency or severity of
relapsing MS attacks; [0244] (e) a decrease in MS clinical
symptoms; [0245] (f) the healing of damaged nerve tissue
(neuro-restoration); [0246] (g) an increase in remyelination of
demyelinated nerves in the central nervous system
(neuro-restoration/protection); [0247] (h) the protection of
damaged nerve tissue from further disease activity
(neuro-protection); [0248] (i) the promotion neuronal outgrowth
(neuro-regeneration) in the central nervous system; [0249] (j) a
decrease in disability caused by MS; [0250] (k) an improvement of
nerve function; and [0251] (l) an enhanced rate of remission.
[0252] In another embodiment the treatment results in a reduction
of one or more clinical symptoms of MS including, but not limited
to loss of sensitivity or changes in sensation such as tingling,
pins and needles or numbness, muscle weakness of variable severity,
very pronounced reflexes, muscle spasms, or difficulty in moving;
difficulties with coordination and balance (ataxia); spasticity;
problems with speech or swallowing, visual problems (nystagmus,
optic neuritis or double vision), fatigue, acute or chronic pain,
facial pain (trigeminal neuralgia), bladder and bowel difficulties,
incontinence, reduced cognitive ability, depression, anxiety and
other emotional abnormalities, sexual dysfunction, Uhthoff's
phenomenon (a worsening of symptoms due to exposure to higher than
usual temperatures), and Lhermitte's sign (an electrical sensation
that runs down the back when bending the neck).
[0253] In one aspect the invention provides a method of
accelerating remission of MS in a subject in need thereof, the
method comprising administering a therapeutically effective amount
of nalfurafine to the subject.
[0254] In one aspect the invention provides a method of
accelerating remission from MS in a subject in need thereof, the
method comprising administering a therapeutically effective amount
of an agent that decreases the level of demyelination in the
subject relative to the level of demyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0255] In one aspect the invention provides a method of
accelerating remission from MS in a subject in need thereof, the
method comprising administering a therapeutically effective amount
of an agent that increases the level of remyelination in the
subject relative to the level of remyelination before administering
the agent, wherein the agent comprises nalfurafine.
[0256] The invention also provides a use of nalfurafine in the
manufacture of a medicament for accelerating remission from MS in a
subject in need thereof.
[0257] The invention also provides nalfurafine for use in
accelerating remission from MS in a subject in need thereof.
[0258] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 5 .mu.g nalfurafine daily, about 0.01 to about 4 .mu.g, about
0.01 to about 3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to
about 2 .mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1
.mu.g, about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5
.mu.g, or about 0.25 .mu.g nalfurafine daily.
[0259] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
[0260] In some embodiments the method comprises administering less
than about 1 .mu.g nalfurafine daily, preferably less than 1 .mu.g
nalfurafine daily.
[0261] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
[0262] The term "enhanced remission of MS" as used herein, means
that the start of the remission process is reached faster and/or
the rate at which remission is achieved is faster (as compared to
subjects not treated with nalfurafine).
[0263] Remission of MS can be measured using any technique known in
the art including but not limited to physical disability status,
biological markers and brain scans using MRI.
[0264] In one aspect the invention provides a method of treating MS
in a human subject in need thereof, the method comprising
administering to the subject about 0.01 to about 5 mg nalfurafine
daily, about 0.05 to about 2.0 mg, about 0.15 to 0.6 mg nalfurafine
daily, wherein the treatment results in one or more clinical
outcomes as compared to subjects not treated with nalfurafine
selected from the group consisting of: [0265] (a) a decrease in MS
disease progression; [0266] (b) a decrease in MS disease severity;
[0267] (c) a decrease in nerve cell demyelination; [0268] (d) a
decrease in frequency or severity of relapsing MS attacks; [0269]
(e) a decrease in MS clinical symptoms; [0270] (f) the healing of
damaged nerve tissue (neuro-restoration); [0271] (g) an increase in
remyelination of demyelinated nerves in the central nervous system
(neuro-restoration/protection); [0272] (h) the protection of
damaged nerve tissue from further disease activity
(neuro-protection); [0273] (i) the promotion neuronal outgrowth
(neuro-regeneration) in the central nervous system; [0274] (j) a
decrease in disability caused by MS; [0275] (k) an improvement of
nerve function; and [0276] (l) an enhanced rate of remission.
[0277] In one aspect the invention provides a method of treating MS
in a human subject in need thereof, the method comprising
administering to the subject about 0.01 to about 5 .mu.g
nalfurafine daily, about 0.01 to about 4 .mu.g, about 0.01 to about
3 .mu.g, about 0.01 to about 2.5 .mu.g, about 0.01 to about 2
.mu.g, about 0.01 to about 1.5 .mu.g, about 0.01 to about 1 .mu.g,
about 0.01 to about 0.75 .mu.g, about 0.01 to about 0.5 .mu.g, or
about 0.25 .mu.g nalfurafine daily, wherein the treatment results
in one or more clinical outcomes as compared to subjects not
treated with nalfurafine selected from the group consisting of:
[0278] (a) a decrease in MS disease progression; [0279] (b) a
decrease in MS disease severity; [0280] (c) a decrease in nerve
cell demyelination; [0281] (d) a decrease in frequency or severity
of relapsing MS attacks; [0282] (e) a decrease in MS clinical
symptoms; [0283] (f) the healing of damaged nerve tissue
(neuro-restoration); [0284] (g) an increase in remyelination of
demyelinated nerves in the central nervous system
(neuro-restoration/protection); [0285] (h) the protection of
damaged nerve tissue from further disease activity
(neuro-protection); [0286] (i) the promotion neuronal outgrowth
(neuro-regeneration) in the central nervous system; [0287] (j) a
decrease in disability caused by MS; [0288] (k) an improvement of
nerve function; and [0289] (l) an enhanced rate of remission.
[0290] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 2.5 .mu.g nalfurafine daily, about 0.025 to about 2 .mu.g,
about 0.05 to about 1 .mu.g, about 0.075 to about 0.75 .mu.g, about
0.1 to about 0.5 .mu.g, or about 0.225 to about 0.325 .mu.g
nalfurafine daily.
[0291] In some embodiments the method comprises administering less
than about 1 .mu.g nalfurafine daily, preferably less than 1 .mu.g
nalfurafine daily.
[0292] In some embodiments the therapeutically effective amount of
nalfurafine to be administered to a human subject is about 0.01 to
about 0.1 .mu.g nalfurafine daily, about 0.025 to about 0.075
.mu.g, about 0.06 to about 0.04 .mu.g, or about 0.05 .mu.g
nalfurafine daily.
[0293] In another aspect the invention provides a method of
treating a demyelinating disease in a subject comprising
identifying a subject who would benefit from a decreased level of
demyelination and administering to the subject a therapeutically
effective amount of an agent that decreases the level of
demyelination relative to the level of demyelination before
administering the agent, wherein the agent comprises
nalfurafine.
[0294] In another aspect the invention provides a method of
increasing remyelination in a subject comprising identifying a
subject who would benefit from an increased level of remyelination
and administering to the subject a therapeutically effective amount
of an agent that increases the level of remyelination relative to
the level of remyelination before administering the agent, wherein
the agent comprises nalfurafine.
[0295] Specifically contemplated as embodiments of the invention
described herein relating to nalfurafine for use in decreasing
demyelination, attenuating demyelination, accelerating remission of
MS, treating MS, treating a demyelinating disease and increasing
remyelination are all of the embodiments of the invention set forth
herein relating to the aspects of the invention that are methods of
decreasing demyelination, attenuating demyelination, accelerating
remission of MS, treating MS, treating a demyelinating disease and
increasing remyelination.
[0296] Additionally, specifically contemplated as embodiments of
the invention described herein relating to the use of nalfurafine
in the manufacture of a medicament for decreasing demyelination,
attenuating demyelination, accelerating remission of MS, treating
MS or for increasing remyelination are all of the embodiments of
the invention set forth herein relating to the aspects of the
invention that are methods of decreasing demyelination, attenuating
demyelination, accelerating remission of MS, treating MS, treating
a demyelinating disease and increasing remyelination.
[0297] In addition, specifically contemplated herein for all
recited method, use and nalfurafine for use aspects of the
invention are all of the embodiments set out herein that relate to
long duration therapy and dosing gaps in long duration therapy.
[0298] The invention consists in the foregoing and also envisages
constructions of which the following gives examples only and in no
way limit the scope thereof.
6.4 Examples
Example 1: Nalfurafine Promotes Functional Recovery from Paralysis
when Administered Therapeutically in the Experimental Autoimmune
Encephalomyelitis (EAE) Model of MS
[0299] Experimental Detail:
[0300] Female, C57BL/6 mice were immunized subcutaneously (s.c.) in
the hind flanks to induce EAE using myelin oligodendrocyte
glycoprotein (MOG) peptide 35-55 (50 mg/mouse) in complete Freund's
adjuvant containing heat-killed Mycobacterium tuberculosis (500
.mu.g/mouse). In addition, pertussis toxin (200 ng/mouse) was
administered intraperitoneally (i.p.) on days 0 and 2. Mice were
weighed and scored daily. On day 17 (vertical dotted line in FIG.
1), mice were started on daily treatment with vehicle only (Veh;
10% tween and 10% DMSO in saline) or nalfurafine at 0.3, 0.1, 0.03,
or 0.01 mg/kg by i.p. injection. Nalfurafine was obtained from the
University of Kansas, Synthetic Chemical Biology Core Laboratory
(97.6% pure by HPLC). Treatment allocation was blinded. The disease
was scored from 0-5 with 0 (normal), 1 (partial tail paralysis), 2
(full tail paralysis), 3 (one hind limb paralysed or severe
disability in both hind limbs), 4 (complete paralysis of both hind
limbs) and 5 (moribund). This model is a standard disease model for
multiple sclerosis and is described in White et al. 2018.
Scientific Reports. 8:259 which is incorporated herein by reference
in its entirety. Shown in FIG. 1 are results combined from 2
independent experiments. **** p<0.0001 &* p<0.05 by
one-way ANOVA with Dunnett's multiple comparison test.
Interpretation and Impact:
[0301] The results demonstrate that nalfurafine is able to treat
on-going disease. The reduction of disease in all
nalfurafine-treated groups indicates recovery from paralysis, which
is complete at some doses (0.1 and 0.03 mg/kg) and unusual in this
model. Finally, the dose at which nalfurafine shows the most rapid
recovery in this example is 0.1 mg/kg with doses above and below
this level appearing less effective.
Example 2: Nalfurafine Reduces Total Disability when Administered
Therapeutically in the EAE Model of MS
Experimental Detail:
[0302] EAE was induced in female C57BL/6 mice as described in
Example 1. On day 17, mice were started on daily treatment with
vehicle only (Veh) or nalfurafine at 0.3, 0.1, or 0.03 mg/kg by
i.p. injection. The area under the curve (AUC) was calculated for
each mouse based upon the daily disease score and represents the
total disability experienced. Shown in FIG. 2 are results from 1
representative experiment. * p<0.05 by one-way ANOVA with
Dunnett's multiple comparison test.
Interpretation and Impact:
[0303] Despite all treatment groups having similar disease scores
at the start of treatment (lower graph), mice treated daily with
nalfurafine had significantly lower total disability by day 45
after immunization to induce EAE (upper graph). Doses of 0.03 and
0.1 mg/kg nalfurafine had the greatest effect at reducing
disability. The 0.1 mg/kg nalfurafine dose results in a 60%
reduction in disease.
[0304] Without wishing to be bound by theory, the inventors believe
that the results in Example 2 highlight the benefits of treatment
with nalfurafine over a period of at least a week. Accordingly, in
some embodiment's administration comprises administration for at
least 7 days, at least 14 days, at least 30 days, at least 45 days,
at least 60 days, at least 120 days, at least 240 days, or at least
360 days.
Example 3: Nalfurafine Promotes Recovery from EAE-Induced Weight
Loss when Administered Therapeutically
Experimental Detail:
[0305] EAE was induced in female C57BL/6 mice as described in
Example 1. Mice were weighed daily and the % change in body weight
calculated. On day 17 (vertical dotted line in FIG. 3), mice were
started on daily treatment with vehicle only (Veh) or nalfurafine
at 0.3, 0.1, or 0.03 mg/kg by i.p. injection.
Interpretation and Impact:
[0306] As shown in FIG. 3, at onset of disease, mice rapidly lose
weight. Once treatment with nalfurafine is initiated (vertical
dotted line), mice recover from EAE-induced weight loss.
Example 4: Nalfurafine Reduces the Immune Cell Infiltration into
the Brain when Administered at Low Doses Therapeutically in the EAE
Model of MS
Experimental Detail:
[0307] EAE was induced in female C57BL/6 mice as described in
Example 1. On day 17, mice were started on daily treatment with
vehicle only (Veh) or nalfurafine at 0.3, 0.1, or 0.03 mg/kg by
i.p. injection. On day 45 after immunization to induce EAE, mice
were culled, and immune cells isolated from the brains. Isolation
was by Percoll gradient as described in White et al. 2018.
Scientific Reports. 8:259. Once isolated, cells were stained with
fluorescently labelled antibodies to identify specific immune cell
types and analysed by flow cytometry. All infiltrating immune cells
were identified by CD45.sup.high expression; CD4 T cells were
identified as CD45.sup.highCD4.sup.+, and macrophages as
CD45.sup.highCD11b.sup.+Gr-1.sup.-. The relative number of cells is
expressed as a ratio to microglia (MG), a brain resident immune
cell identified as CD45.sup.mediumCD11b.sup.+. * p<0.05 by
one-way ANOVA with Dunnett's multiple comparison test.
Interpretation and Impact:
[0308] As shown in FIG. 4, at day 45, there was a significant
elevation in immune cells in the brains of vehicle-treated EAE mice
compared to healthy animals. Treatment with 0.03 mg/kg nalfurafine
significantly reduced the number of infiltrating immune cells
suggesting that at this dose, nalfurafine can have immunomodulatory
properties. Interestingly, while mice treated with 0.1 nalfurafine
had similar levels of infiltrating cells as vehicle-treated
animals, these mice had no overt signs of disease and had recovered
fully from paralysis (FIG. 1).
Example 5: Nalfurafine Promotes Functional Recovery from Paralysis
when Administered Before the Onset of Paralysis in the EAE Model of
MS
Experimental Detail:
[0309] EAE was induced in female C57BL/6 mice as described in
Example 1. On day 17 (vertical dotted line in FIG. 5), mice were
started on daily treatment with vehicle only (Veh) or nalfurafine
at 0.3, 0.1, or 0.03 mg/kg by i.p. injection. Shown in FIG. 5 are
results in mice that were not sick at the time of treatment but
developed disease later. * p<0.05 by two-way ANOVA with
Holm-Sidak's multiple comparison test.
Interpretation and Impact:
[0310] Treating with nalfurafine prior to disease onset did not
alter the onset of disease. However, treatment with nalfurafine led
to a rapid recovery from paralysis compared to vehicle-treated
mice. These data suggest that treating with nalfurafine will also
be effective at reducing total disability if administered before
disease but may not prevent onset.
Example 6: Myelination is Improved in Mice Treated with Nalfurafine
after the Onset of Paralysis in the EAE Model of MS
Experimental Detail:
[0311] EAE was induced in female C57BL/6 mice as described in
Example 1. On day 17, mice were started on daily treatment with
vehicle only or nalfurafine at 0.03 mg/kg by i.p. injection. On day
45 after immunization to induce EAE, mice were culled, and spinal
cords were processed for transmission electron microscopy (TEM).
Shown in FIG. 6 are representative TEM images of spinal cord
sections from a healthy (A), vehicle-treated EAE (B), or
nalfurafine-treated EAE mouse (C) stained to show that dark myelin
rings around the nerve axons.
Interpretation and Impact:
[0312] At day 45, there was a significant reduction in the dark
stained myelin in the spinal cord of the vehicle-treated EAE mice
suggesting demyelination has occurred. Additionally, the nerve
axons appear bloated and the cytoplasm disorganized suggesting
cellular stress. In contrast, the nerve axons appear healthy and
well-myelinated in the nalfurafine-treated mouse, which is
concordant with full functional recovery.
Example 7: Nalfurafine Improved Weight Gain when Administered after
Demyelination in the Cuprizone Model of Demyelination
Experimental Detail:
[0313] Female, C57BL/6 mice were fed 0.3% cuprizone in the diet for
5 weeks to induce demyelination. At the start of week 4 (vertical
dashed line in FIG. 7), mice were started on daily treatment with
vehicle only or nalfurafine 0.1 mg/kg by i.p. injection. At the
start of week 5 (vertical dotted line in FIG. 7), cuprizone was
removed from the diet to enable spontaneous remyelination. Mice
were weighed daily and the % weight change calculated.
Interpretation and Impact:
[0314] As shown in FIG. 7, cuprizone caused significant weight loss
in mice as previously reported. This weight loss was reversed
significantly more effectively by administration of nalfurafine
than vehicle alone.
Example 8: Nalfurafine Enhances the Functional Recovery of
Coordination and Balance when Administered after Demyelination in
the Cuprizone Model of Demyelination
Experimental Detail:
[0315] Female, C57BL/6 mice were fed 0.3% cuprizone in the diet for
5 weeks to induce demyelination and treated with nalfurafine as
described in Example 7. Behavioural tests including the rotarod
assay, which measures coordination, were performed weekly. Mice
were trained on an accelerating rotarod apparatus (Panlab, Harvard
Apparatus) over a period of 4 to 5 days before recording baseline
latencies at day 0 followed by weekly measurements throughout
cuprizone treatment and recovery. The rotarod was set to 4
rotations per minute (rpm) and an acceleration rate of 40 rpm with
a maximum cut-off time of 5 minutes. The time and speed at which
the animal falls off the rotating rod was recorded and the average
of 3 replicates recorded. Data shows performance at week 9
following Veh or nalfurafine (0.1 mg/kg) treatment relative to
performance at week 5. * p<0.05 by Students t-test.
Interpretation and Impact:
[0316] As shown in FIG. 8, cuprizone impaired coordination in mice
as previously reported. Cuprizone-induced disability was reversed
by administration of nalfurafine. These data suggest that
nalfurafine is effective at reducing disability in a model of
non-immune mediated demyelination such as that found in some
progressive MS patients.
Example 9: Nalfurafine Enhances Myelination when Administered after
Demyelination in the Cuprizone Model of Demyelination
Experimental Detail:
[0317] Female, C57BL/6 mice were fed 0.3% cuprizone in the diet for
5 weeks to induce demyelination as described in Example 7. On day
65, mice were culled, and brains were processed for transmission
electron microscopy (TEM). Shown are representative TEM images of
sections from the corpus callosum of a healthy (no cuprizone),
vehicle-treated & cuprizone-treated, or nalfurafine-treated
& cuprizone-treated mouse stained to show the dark myelin rings
around the nerve axons. Myelin was quantified by g-ratio, which is
the inner axonal diameter divided by the total outer diameter.
Interpretation and Impact:
[0318] As shown in FIG. 9, cuprizone caused a loss of myelin and a
concurrent disruption in regular axonal structures in the corpus
callosum compared to healthy controls. In contrast, more myelin was
detected, and the structure was less disorganized in the corpus
callosum of animals treated with cuprizone & nalfurafine. These
data indicate that nalfurafine treatment promotes remyelination and
repair after cuprizone-induced, non-immune-mediated demyelination.
A similar non-immune associated demyelination occurs in some
progressive MS patients.
Example 10: Nalfurafine Promotes Functional Recovery from Paralysis
when Administered Therapeutically in the Experimental Autoimmune
Encephalomyelitis (EAE) Model of MS
Experimental Detail:
[0319] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 10. On the day of disease
onset (score.gtoreq.1, dotted line), mice were started on daily
treatment with vehicle only (Veh) or nalfurafine at 0.3, 0.1, 0.03,
0.01, or 0.003 mg/kg by i.p. injection. Treatment allocation was
blinded. Shown are the aligned scores from mice (n=33 in Veh, 3 in
0.3, 4 in 0.1, 5 in 0.03, 20 in 0.01, and 4 in 0.003 mg/kg groups)
starting from onset/treatment initiation. One animal in the 0.3
mg/kg nalfurafine group and 2 from the vehicle group were
euthanized at day 17-18. ****p<0.0001 by two-way ANOVA all doses
(except 0.3 mg/kg) compared to vehicle.
Interpretation and Impact:
[0320] By treating after the onset of disease (paralysis), we show
that nalfurafine is able to treat on-going disease. The reduction
of disease in all nalfurafine-treated groups indicates recovery
from paralysis, which is complete at some doses (0.01 and 0.03
mg/kg); full recovery from disease is unusual in this model and the
efficacy of the nalfurafine treatment is surprising. Finally, the
dose at which nalfurafine shows the most rapid recovery in this
example is 0.01 mg/kg, and this finding has been replicated in 6
independent experiments.
Example 11: Nalfurafine is not Effective when Administered as a
Short 4-Day Course Starting at Disease Onset in EAE Model of MS
Experimental Detail:
[0321] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 11. On the day of disease
onset (score.gtoreq.1, dotted line), mice were started on daily
treatment with vehicle only or nalfurafine at 0.01 mg/kg by i.p.
injection daily throughout the experimental course or only for four
days (shaded area). Shown are the aligned scores from mice
(n=5/group) starting from onset/treatment initiation. **p<0.01
by two-way ANOVA NaIF (full treatment) compared to nalfurafine (4
days) or vehicle.
Interpretation and Impact:
[0322] Treatment with nalfurafine does not enhance recovery when
administered for only four days starting from disease onset,
whereas treatment with a longer duration does enhance recovery
effectively.
Example 12: Nalfurafine does not Alter Peak Disease when
Administered Therapeutically in the EAE Model of MS
Experimental Detail:
[0323] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 12. On the day of disease
onset (score.gtoreq.1), mice were started on daily treatment with
vehicle only or nalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003 mg/kg
by i.p. injection. The peak disease score during the first EAE
episode was recorded and shown are the mean and standard error of
individual mice (n=33 in Veh, 3 in 0.3, 4 in 0.1, 5 in 0.03, 20 in
0.01, and 4 in 0.003 mg/kg groups). No significant differences were
found between any nalfurafine dose and vehicle by Kruskal-Wallis
with Dunn's multiple comparison test. These data are from the same
experiments as Example 10.
Interpretation and Impact:
[0324] Because no difference in peak disease score was found at any
dose of nalfurafine compared to vehicle, nalfurafine did not appear
to alter the initial immune-mediated neuroinflammatory event that
leads to demyelination and paralysis. This finding suggests that
the functional improvement observed (i.e. the recovery from
paralysis) occurs because the initial insult has been repaired and
perhaps not because the initial insult itself was stopped.
Example 13: Nalfurafine Promotes Full Recovery from EAE-Induced
Paralysis when Administered Therapeutically
Experimental Detail:
[0325] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 13. On the day of disease
onset (score.gtoreq.1), mice were started on daily treatment with
vehicle only or nalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003 mg/kg
by i.p. injection. Mice were considered recovered if they received
a score.ltoreq.0.5 by day 23 post treatment initiation. Shown are
the percentages of mice in each group that recovered (n=33 in Veh,
3 in 0.3, 4 in 0.1, 5 in 0.03, 20 in 0.01, and 4 in 0.003 mg/kg
groups). ****p<0.0001, **p<0.01, and *p<0.05 by Fisher's
exact test. These data are from the same experiments as Example
10.
Interpretation and Impact:
[0326] Treatment with nalfurafine enables full functional recovery
(i.e. no paralysis) when administered therapeutically and at a wide
range of doses (0.003-0.1 mg/kg all show a significant effect).
Full recovery in this model of disease is unusual. The efficacy
achieved with the treatment of nalfurafine is extraordinary.
Example 14: Nalfurafine Promotes Full Recovery from EAE-Induced
Paralysis when Administered Therapeutically with an EC.sub.50 of
<0.001 Ma/Kg
Experimental Detail:
[0327] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 14. On the day of disease
onset (score.gtoreq.1), mice were started on daily treatment with
vehicle only or nalfurafine at 0.1, 0.03, 0.01, or 0.003 mg/kg by
i.p. injection. Mice were considered recovered if they received a
score.ltoreq.0.5 by day 23 post treatment initiation. Shown are the
percentages of mice in each group that recovered (n=33 in Veh, 4 in
0.1, 5 in 0.03, 20 in 0.01, and 4 in 0.003 mg/kg groups). A
dose-response curve has been fitted from a dose of 0.1 mg/kg, in
which 100% recovered, to the vehicle alone, in which 12.1%
recovered. This curve calculates an EC.sub.50 of <0.001 mg/kg.
These data are from the same experiments as Example 13.
Interpretation and Impact:
[0328] Treatment with Nalfurafine enables full functional recovery
(i.e. no paralysis) when administered therapeutically and at a wide
range of doses (0.003-0.1 mg/kg all show a significant effect).
Full recovery in this model of disease is unusual. The efficacy
achieved with the treatment of nalfurafine is extraordinary. To
achieve 50% of this effect (i.e. EC.sub.50) an estimated dose of
<0.001 mg/kg is required.
Example 15: Nalfurafine Promotes Sustained Functional Recovery from
EAE-Induced Paralysis when Administered Therapeutically
Experimental Detail:
[0329] EAE was induced in female C57BL/6 mice as described in
Example 1,
[0330] Results are shown in FIG. 15. On the day of disease onset
(score.gtoreq.1), mice were started on daily treatment with vehicle
only or nalfurafine at 0.3, 0.1, 0.03, 0.01, or 0.003 mg/kg by i.p.
injection. Mice were considered recovered if they received a
score.ltoreq.0.5 by day 23 post treatment initiation. Shown are the
number of days mice were in recovery in each group (n=33 in Veh, 3
in 0.3, 4 in 0.1, 5 in 0.03, 20 in 0.01, and 4 in 0.003 mg/kg
groups). ****p<0.0001, **p<0.01, and *p<0.05 by one-way
ANOVA with Holm-Sidak's multiple comparison test. These data are
from the same experiments as Example 10.
Interpretation and Impact:
[0331] Treatment with nalfurafine enables a sustained functional
recovery (i.e. no paralysis) when administered therapeutically and
at a wide range of doses (0.003-0.1 mg/kg all show a significant
effect).
Example 16: Nalfurafine Promotes Functional Recovery from Paralysis
in Male Mice when Administered Therapeutically in EAE Model of
MS
Experimental Detail:
[0332] EAE was induced in male C57BL/6 mice as described in Example
1. Results are shown in FIG. 16. On the day of disease onset
(score.gtoreq.1, line), mice were started on daily treatment with
vehicle only or nalfurafine at 0.01 mg/kg by i.p. injection.
[0333] Treatment allocation was blinded. Shown are the aligned
scores from mice (n=5/group) starting from onset/treatment
initiation. ****p<0.0001 by two-way ANOVA compared to
vehicle.
Interpretation and Impact:
[0334] Nalfurafine is effective at enabling functional recovery
from paralysis in both females and males.
Example 17: Nalfurafine Promotes Full Recovery in Male Mice when
Administered Therapeutically in EAE Model of MS
Experimental Detail:
[0335] EAE was induced in male C57BL/6 mice as described in Example
1. Results are shown in FIG. 17. On the day of disease onset
(score.gtoreq.1), mice were started on daily treatment with vehicle
only or nalfurafine at 0.01 mg/kg by i.p. injection. Mice were
considered recovered if they received a score.ltoreq.0.5 by day 23
post treatment initiation. Shown are the percentages of mice in
each group that recovered (n=5/group). **p<0.01 by Fisher's
exact test. These data are from the same experiments as Example
16.
Interpretation and Impact:
[0336] Treatment with nalfurafine promotes full recovery (i.e. no
paralysis) in both female and male when administered
therapeutically.
Example 18: Nalfurafine Promotes Sustained Recovery in Male Mice
from EAE-Induced Paralysis when Administered Therapeutically
Experimental Detail:
[0337] EAE was induced in male C57BL/6 mice as described in Example
1. Results are shown in FIG. 18. On the day of disease onset
(score.gtoreq.1), mice were started on daily treatment with vehicle
only or nalfurafine at 0.01 mg/kg by i.p. injection. Mice were
considered recovered if they received a score.ltoreq.0.5 by day 23
post treatment initiation. Shown are the number of days mice were
in recovery in each group (n=5/group). ****p<0.0001 by Student's
t test. These data are from the same experiments as Example 16.
Interpretation and Impact:
[0338] Treatment with nalfurafine enables a sustained functional
recovery (i.e. no paralysis) in both females and males when
administered therapeutically.
Example 19: Nalfurafine Treatment Reduces the Immune Cell
Infiltration into the Brain when Administered Therapeutically in
the EAE Model of MS (A) Whereas U-50488 does not (B)
Experimental Detail:
[0339] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIGS. 19A and 19B. On the day of
disease onset (score.gtoreq.1), mice were started on daily
treatment with vehicle only or nalfurafine at 0.3, 0.1, 0.03, 0.01,
or 0.003 mg/kg by i.p. injection (A). In a separate experiment,
mice were similarly treated with vehicle alone or U-50488, a KOR
agonist at 1.6 and 5 mg/kg (B). During the chronic phase (>24
days post treatment initiation), mice were culled, and immune cells
isolated from the brains. Isolation was by Percoll gradient as
described in White et al. 2018. Scientific Reports. 8:259. Once
isolated, cells were stained with fluorescently labelled antibodies
to identify specific immune cell types and analysed by flow
cytometry. All infiltrating immune cells were identified by
CD45.sup.high expression. The relative number of cells is expressed
as a ratio to microglia (MG), a brain resident immune cell
identified as CD45.sup.mediumCD11b.sup.+.*p<0.05 by one-way
ANOVA with Holm-Sidak's multiple comparison test compared to
vehicle. NS, not-significant.
Interpretation and Impact:
[0340] In the chronic stage of EAE, there was a significant
elevation in immune cells in the brains of vehicle-treated EAE mice
compared to healthy animals (A). Treatment with 0.03 and 0.01 mg/kg
nalfurafine significantly reduced the number of infiltrating immune
cells suggesting that at these doses, nalfurafine can have
immunomodulatory properties. Interestingly, while mice treated with
0.1 and 0.003 nalfurafine had similar levels of infiltrating cells
as vehicle-treated animals, these mice had no overt signs of
disease and had recovered fully from paralysis (FIG. 13).
Additionally, nalfurafine but not U-50488 reduced neuroinflammation
in this model indicating that not all KOR agonists have this
activity (B).
Example 20: Myelination is Improved in Mice Treated with
Nalfurafine after the Onset of Paralysis in the EAE Model of MS
Experimental Detail:
[0341] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 20. On the day of disease
onset (score.gtoreq.1), mice were started on daily treatment with
vehicle only or nalfurafine at 0.03 or 0.01 mg/kg by i.p.
injection. During the chronic phase (>24 days post treatment
initiation), mice were culled, perfused with 4% paraformaldehyde
and spinal cords were processed for histology. Sections were
stained with luxol fast blue to assess the % area of the spinal
cord that is demyelinated (i.e. does not stain with luxol fast
blue). % demyelination was assessed using ImageJ. Shown are the
means and standard error of individual values from vehicle (n=7) or
0.01 (n=6) and 0.03 (n=4) nalfurafine-treated EAE mice. **p<0.01
by one-way ANOVA with Holm-Sidak's multiple comparison test.
Interpretation and Impact:
[0342] During the chronic phase, when nalfurafine enabled full
functional recovery in mice, there was a significant reduction in
the percentage of demyelination in the spinal cord of the
nalfurafine-treated EAE mice suggesting remyelination may have
occurred.
Example 21: Nalfurafine does not Alter the Proportion of Major
Lymphocyte Populations in the Spleen During the Chronic Phase of
EAE
Experimental Detail:
[0343] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 21. On the day of disease
onset (score.gtoreq.1), mice were started on daily treatment with
vehicle only or nalfurafine at 0.01 mg/kg by i.p. injection. During
the chronic phase (27 days post treatment initiation), mice were
culled and their splenocytes assessed by flow cytometry. The
percentage of the major lymphocyte populations were identified
using CD4 (CD4 T helper cells), CD8 (CD8 cytotoxic T cells), and
B220 (B cells), and expressed as % live leukocytes (i.e. CD45+
cells). Shown are the means and standard error of individual mice
with n=3 (healthy), 4 (vehicle) and 8. No significant differences
were found between vehicle and healthy or nalfurafine by one-way
ANOVA with Holm-Sidak's multiple comparison test.
Interpretation and Impact:
[0344] Nalfurafine do not alter the proportion of the major
lymphocyte populations in the spleen despite reducing the number of
infiltrating immune cells into the central nervous system. The
maintenance of normal lymphocyte numbers in the spleen in the
nalfurafine treated mice indicates that nalfurafine does not reduce
immune cell infiltration into the brain by killing immune
cells.
Example 22: Nalfurafine does not Alter the Overall Number of CD4 T
Helper Cells in the Spleen but Shifts the CD4 T Cells from an
Effector to Memory State being Suggestive of Immune Resolution
During the Chronic Phase of EAE
Experimental Detail:
[0345] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 22. On the day of disease
onset (score.gtoreq.1), mice were started on daily treatment with
vehicle only or nalfurafine at 0.01 mg/kg by i.p. injection. During
the chronic phase (27 days post treatment initiation), mice were
culled and their splenocytes assessed by flow cytometry. Naive CD4
T cells (CD4.sup.+CD44.sup.-CD62L.sup.high), effector CD4 T cells
(CD4.sup.+CD44.sup.+CD62L.sup.-), and central memory CD4 T cells
(CD4.sup.+CD44.sup.+CD62L.sup.high) are expressed as % CD4 T cells.
"Teff:cm ratio" is the ratio of effector to central memory T cells.
Shown are the means and standard error of individual mice with n=3
(healthy), 4 (vehicle) and 8. **p<0.01 and *p<0.05 by one-way
ANOVA with Holm-Sidak's multiple comparison test.
Interpretation and Impact:
[0346] The increased effector to central memory ratio in the
vehicle-treated mice with EAE compared to healthy mice indicates an
on-going and active immune response mediated by CD4 T cells. The
overall number of CD4 T cells was the same between nalfurafine and
vehicle treated mice. The reduced ratio in the nalfurafine-treated
compared to the vehicle-treated mice indicates a shift toward a
memory phenotype which occurs during the resolution phase of the
immune response. The shift to a memory state indicates that immune
resolution is occurring in nalfurafine-treated mice in a model of
MS where disease is driven by an active immune response.
Example 23: Nalfurafine Reduces Disease but does not Enable Full
Recovery when the Kappa Opioid Receptor (KOR) is Blocked
Experimental Detail:
[0347] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 23. On the day of disease
onset (score>1, dotted line), mice were treated with vehicle
only (daily), nalfurafine (0.01 mg/kg by i.p. injection daily), the
KOR antagonist norBNI (10 mg/kg by i.p. injection weekly), or both
nalfurafine and norBNI. Shown are the aligned scores from mice
(n=8-9/group) starting from onset/treatment initiation.
****p<0.0001 by two-way ANOVA NaIF compared to vehicle or
NaIF+noBNI.
Interpretation and Impact:
[0348] Administration of the KOR antagonist, norBNI, abolishes the
ability of nalfurafine to enable full recovery from paralysis (i.e.
score<0.5), and this finding indicates that the KOR is required
for the full effect of nalfurafine. The finding that nalfurafine is
effective at reducing disease independently of the KOR (i.e. in the
presence of norBNI) indicates that the full mechanism by which
nalfurafine exerts its effects is more complex than KOR
activation.
Example 24: Activation of the KOR is Required for Full Recovery
from Paralysis Mediated by Nalfurafine
Experimental Detail:
[0349] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 24. On the day of disease
onset (score>1, dotted line), mice were treated with vehicle
only (daily), nalfurafine (0.01 mg/kg by i.p. injection daily), the
KOR antagonist norBNI (10 mg/kg by i.p. injection weekly), or both
nalfurafine and norBNI. The peak disease score during the first EAE
episode was recorded, and mice were considered recovered if they
received a score<0.5 by day 23 post treatment initiation. Shown
are the peak disease scores, the percentage of mice in each group
that recovered, and the number of days in recovery (n=8-9/group).
**p<0.01 and ****p<0.0001 by Fisher's exact test (%
recovered) or one-way ANOVA with Holm-Sidak's multiple comparison
test (# days in recovery). These data are from the same experiments
as Example 23.
Interpretation and Impact:
[0350] Administration of the KOR antagonist, norBNI, abolishes the
ability of nalfurafine to enable and sustain recovery from
paralysis (i.e. score<0.5), and this finding indicates that the
KOR is required for the full effect of nalfurafine at promoting
full recovery but not disease reduction.
Example 25: Myelination is Improved in Mice Treated with
Nalfurafine after the Onset of Paralysis in the EAE Model of MS
Experimental Detail:
[0351] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 25. On the day of disease
onset (score.gtoreq.1), mice were started on daily treatment with
vehicle only or nalfurafine 0.01 mg/kg by i.p. injection. During
the chronic phase (>24 days post treatment initiation), mice
were culled, perfused with 4% paraformaldehyde and spinal cords
were processed for histology. Sections were stained with luxol fast
blue to assess demyelination. The region of interest taken for
analysis is shown in 25A. Note the presence of demyelinated regions
(lesions) with less luxol fast blue (LFB) staining (myelin) in the
ventral horn in EAE mice receiving vehicle (circle--25B) and no
demyelinated lesions present in mice treated with nalfurafine
(25C). Quantified data is shown in 25D. For each image, 5
randomised regions of the ventral horn of the spinal cord were
analysed in ImageJ using mean grey value and integrated pixel
density as an indicator of myelin density. Data is from two
individual experiments with n=4 (vehicle), n=8 (nalfurafine) EAE
mice respectively. Scale bar=50 .mu.m. *p<0.05 by students
t-test.
Interpretation and Impact:
[0352] EAE disease induces extensive lesions in the spinal cord
(see vehicle only (25B), characterised by a loss of myelin and
neurodegeneration, demonstrating that EAE is a destructive disease
in the CNS. Treatment of this disease state with nalfurafine
reduces this lesion load and demyelination, suggesting that
treatment restores the spinal cord tissue to a near normal state by
remyelination.
Example 26: Nalfurafine Treatment Decreases Cellular Infiltration
into the Spinal Cord when Administered Therapeutically in the EAE
Model of MS
Experimental Detail:
[0353] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 26. On the day of disease
onset (score>1), mice were started on daily treatment with
vehicle only or nalfurafine 0.01 mg/kg by i.p. injection. During
the chronic phase (>24 days post treatment initiation), mice
were culled, perfused with 4% paraformaldehyde and spinal cords
were paraffin embedded for histology. 10 .mu.M coronal sections
were stained with Hematoxylin and Eosin (H&E) to assess of
leucocyte infiltration, a marker of inflammation within lesions
induced in EAE disease. Note the large number of leucocytes present
in the ventral horn of vehicle treated EAE mice, than in EAE mice
administered nalfurafine. Images were scored by a blinded observer
for the level of infiltration on a scale ranging from 0 (no
infiltration) to 3 (maximum infiltration). Data is from two
individual experiments: n=7 mice (11 sections) for EAE Vehicle; and
n=9 mice (13 sections) for EAE mice treated with nalfurafine. Scale
bar=50 .mu.m). Students t-test, *p<0.05.
Interpretation and Impact:
[0354] EAE disease induces substantial histopathology in the spinal
cord. H&E staining of leucocytes is an indicator of lesion
severity, with the higher number of infiltrating cells, the more
severe the lesion, including demyelination, as shown in the vehicle
only panel and by quantification. Treatment with nalfurafine shows
a surprising reduction of infiltrating leucocytes, with a near
absence of lesions and demyelination indicating that treatment may
resolve lesions and/or cause remyelination.
Example 27: Nalfurafine Treatment Reduces the Level of Activated
Astrocytes in the Spinal Cord when Administered Therapeutically in
the EAE Model of MS
Experimental Detail:
[0355] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 27A-B. On day 17, mice were
started on daily treatment with vehicle only or nalfurafine at 0.01
mg/kg by i.p. injection. On day 45 after immunization to induce
EAE, mice were culled, and spinal cords were processed for
immunohistochemistry (IHC). Shown in FIG. 27A are representative
glial fibiliary acid protein (GFAP) immunolabeled cells (black
staining) from coronal sections of the ventral horn of the spinal
cord taken from EAE mice. The images are from 10 .mu.M paraffin
embedded sections, stained with Rabbit anti-GFAP at (1:1000, DAKO)
before being photographed at 20.times. magnification, scale bar=50
.mu.m. The number of astrocytes per section in a standard ROI were
counted using the cell counter plug-in in ImageJ. Two sections were
assessed per animal. Sections assessed n=7 (10-13 sections, from 2
individual experiment. ***p=0.0003 (FIG. 27B).
Interpretation and Impact:
[0356] As shown and quantified in FIG. 27A, at day 45, there was
significant elevation in the activated GFAP.sup.+ astrocytes in the
spinal cord of vehicle treated EAE mice. Astrocytes are recognized
to be early and highly active players during lesion formation and
key for providing peripheral immune cells access to the central
nervous system (Ponath et al. The Role of Astrocytes in Multiple
Sclerosis. Front Immunol. 2018; 9: 217). Treatment with 0.01 mg/kg
i.p. nalfurafine significantly reduces the number of activated
astrocytes suggesting that nalfurafine treatment can have a
neuroprotective and anti-inflammatory effect on the spinal cord
tissue in the disease state (FIG. 27B).
Example 28: Nalfurafine Treatment Enhances Recovery from Weight
Loss when Administered Therapeutically in the Cuprizone
Demyelination Disease Model of MS
[0357] FIG. 28A shows a time course of cuprizone induced
demyelination and treatment regime.
Experimental Details:
[0358] A demyelinating disease state was induced in female C57BL/6
mice (8-14 weeks old and between 17-23 grams in weight). As shown
in the timeline of FIG. 28A, the mice were fed cuprizone-containing
chow (0.3% (w/w) cuprizone) or chow only (normal controls) for 35
days, at which point they were switched back to standard chow. At
day 28, mice were started on daily treatment with vehicle only
(DMSO: Tween 80: Saline) or nalfurafine at 0.1 mg/kg by i.p.
injection or U-50488 at 1.6 mg/kg by i.p. injection. On day 70,
mice were culled and brain tissue were processed for transmission
electron microscopy (TEM). Mice were weighed daily and the % weight
change calculated.
Interpretation and Impact:
[0359] This model is well established as a tool for the study of
non-immune system induced demyelination. This model enables the
assessment of putative remyelination-promoting therapeutics
(Matsushima and Morell, 2001. The neurotoxicant, cuprizone, as a
model to study demyelination and remyelination in the central
nervous system. Brain Pathol. 11, 107-116).
[0360] FIG. 28B shows cuprizone induced weight change over the time
course of study.
Experimental Details:
[0361] A demyelinating disease state was induced in female C57BL/6
mice as described in Example 28 and illustrated in FIG. 28.
Interpretation and Impact:
[0362] Mice treated with 0.3% cuprizone (CPZ) lose weight as the
disease is induced, compared to mice with normal diet,
corresponding to disease induction and severity.
[0363] FIG. 28C shows that nalfurafine treatment enhances weight
gain in the recovery phase of the cuprizone demyelination disease
model of MS, whereas U-50488 does not.
Experimental Details:
[0364] A demyelinating disease state was induced in female C57BL/6
mice as described in FIG. 28C. Diseased animals were treated with
Vehicle only, nalfurafine (0.1 mg/kg), U-50488 (1.6 mg/kg) as
described in FIG. 28A. Mice were weighed daily and the % weight
change calculated. *p<0.05=nalfurafine treated mice;
#p<0.05=U-50488 treated mice. Two-way repeated measures ANOVA,
followed by Turkey's multiple comparison tests. (n=15 mice/group
from 3 experimental replicates. ANOVA revealed a significant
interaction F(40, 600)=2.212 (p<0.0001) with significant time
F(8, 600)=101.2 (p<0.0001) and treatment effects F(5.75)=5.52
(P<0.0002).
Interpretation and Impact:
[0365] Mice treated with 0.3% cuprizone (CPZ) lose weight as the
disease is induced. Mice recover when returned to normal chow
(removal of cuprizone) (FIG. 28C). Treatment with nalfurafine
enhances recovery of the lost weight faster compared to mice with
vehicle only or treatment with U-50488.
FIG. 29: Nalfurafine Treatment Enhances Remyelination when
Administered after Demyelination in the Cuprizone Demyelination
Disease Model of MS
Experimental Details:
[0366] A demyelinating disease state was induced in female C57BL/6
mice as illustrated in FIG. 28A. The results are shown in FIG.
29A-G. Panels A-D of FIG. 29 show representative Transmission
Electron Microscopy (TEM) images of the corpus callosum of mice (A)
fed normal diet and (B-D) fed 0.3% cuprizone to induce
demyelination. Following the time course shown in FIG. 28A,
cuprizone fed mice were administered (B) vehicle only treatment,
(C) U-50488 (1.6 mg/kg/i.p.) and (D) nalfurafine (0.1 mg/kg/i.p.)
and then sacrificed on experimental day 70. Scale bars represent
2000 nm.
[0367] FIG. 29 (E) shows the quantification and analysis of the
g-ratios shows a significant difference between treatment groups
F(3,953)=21.18 (p<0.0001). Mice fed a normal diet have a mean
g-ratio of 0.78.+-.0.09 in contrast to mice fed 0.3% cuprizone that
have a significant increase in g-ratio of 0.84.+-.0.1 corresponding
to the decreased myelin thickness (####p<0.0001). Mice fed a
diet with cuprizone treated with nalfurafine (0.1 mg/kg/i.p.)
(0.75.+-.0.15) show a significant reduction in g-ratio compared to
Vehicle treated controls (****p<0.0001), corresponding to an
increased myelin thickness. Mice fed a diet with cuprizone treated
with U-50488 show a somewhat increased myelin thickness compared to
vehicle-treated controls with a mean g-ratio of (0.80)
(**p<0.01), but, surprisingly, nalfurafine treatment showed a
significant increase in myelin thickness (decrease in g-ratio)
compared to mice treated with U-50488 (1.6 mg/kg/i.p.) ({circumflex
over ( )}{circumflex over ( )}{circumflex over ( )}p<0.001),
indicating that nalfurafine is significantly more effective at
increasing myelin thickness than U-50488. Data represents
measurements of 5 TEM images of the corpus callosum from two-three
mice per treatment group and g-ratios calculated (a measure of
myelin thickness) using Image J software. Analysis was performed by
individuals blinded to treatment groups. (n=204-267 axons per
treatment group).
[0368] FIG. 29 (F) shows the quantification and analysis of the
number of myelinated axons vs non-myelinated axons in a region of
interest (390 .mu.m.sup.2). n=20 images per treatment group (from
n=2-3 mice).
[0369] FIG. 29 (G) shows the auantification and analysis of the
area of myelin staining per TEM image was performed using Image J
software (20 images per treatment from n=2-3 mice sacrificed on day
70). TEM images were colour inverted (myelin white) and a threshold
used to reveal myelin. The area of this myelin threshold measured
for each treatment group.
[0370] All data analysed by one-way ANOVA followed by Turkeys
multiple comparisons test. Significant differences compared to
vehicle only are depicted by *; between normal mice and
cuprizone/vehicle treated mice #; and between nalfurafine and
U-50488 by {circumflex over ( )}. (*p<0.05; **p<0.01;
***p<0.001; ****P<0.0001).
Interpretation and Impact:
[0371] As shown in FIG. 29A-G, demyelination was very apparent in
the corpus collosum of the brain of cuprizone-induced, vehicle only
treated animals (Panel B). The ratio between axonal circumference
and myelin circumference (g-ratio) decreases with normal
myelination. The cuprizone induced animals treated with nalfurafine
show a more normal axonal-myelin structure, the myelinated axons
are densely packed within white matter and the myelin sheaths of
neighboring fibers often directly touch. The staining of the myelin
sheaths (black) is more prominent indicating increased
remyelination. Ultrastructurally, this nalfurafine tissue is
surprisingly similar to that of the naive (normal) tissue.
Quantitatively, the nalfurafine tissue has a significantly lower
g-ratio compared to vehicle only treated indicative of enhanced
remyelination, with a g-ratio closer to that of naive (normal)
tissue. This is further supported by analysis of the percentage
increase in the number of myelinated axons and percentage increase
in area of myelination in the nalfurafine treated animals. In
contrast, treatment with the compound U-50488 did not show repair
or restoration to a near normal state. Qualitatively, the
axonal-myelin structure is disorganised, there is a loss of axons,
and overtly enlarged axonal-myelin structures. Quantitatively,
U-50488 treatment has outcomes similar to that of the vehicle only
treated samples (i.e. no discernible remyelination), whereas
nalfurafine treatment shows similar outcomes to the naive tissue.
Qualitatively and quantitatively, nalfurafine treatment enhances
remyelination that is indicative of a near-full recovery following
a demyelination insult of cuprizone.
Example 30: Nalfurafine is More Effective at Promoting Functional
Recovery than Clemastine Fumarate, a Known Remyelinating Drug
Experimental Detail:
[0372] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 30. On the day of disease
onset (score>1, dotted line), mice were treated with vehicle
only (daily, n=9)) or nalfurafine (0.01 mg/kg by i.p. injection
daily; n=8). In a separate similar experiment, mice were treated
with vehicle only (n=5) or clemastine fumarate (10 mg/kg by i.p.
injection; n=7). Shown are the aligned scores from mice starting
from onset/treatment initiation. ****p<0.0001 by two-way ANOVA
NaIF or clemastine compared to vehicle.
Interpretation and Impact:
[0373] Clemastine fumarate, an anti-histamine which also
antagonizes the muscarinic receptor, has been shown to reduce
chronic disability in the EAE model when used at 10 mg/kg starting
at the time of immunization. Additionally, it has been shown to
enhance remyelination in mice and humans (Li et al. 2015,
Clemastine rescues behavioral changes and enhances remyelination in
the cuprizone mouse model of demyelination. Neurosci Bull.; 31:
617-625; Green, A. J et al., 2017 Clemastine fumarate as a
remyelinating therapy for multiple sclerosis (ReBUILD): a
randomised, controlled, double-blind, crossover trial. Lancet Lond.
Engl. 390, 2481-2489). In our EAE model, clemastine is similarly
effective to previously published reports, but is much less
effective than nalfurafine at enabling full functional recovery
(Mei, F. et al. 2016, Accelerated remyelination during inflammatory
demyelination prevents axonal loss and improves functional
recovery. ELife 5). This example shows that nalfurafine is superior
to clemastine fumarate in this model.
Example 31: Nalfurafine Promotes a Greater and More Sustained
Recovery than Clemastine Fumarate, a Known Remyelinating Drug
Experimental Detail:
[0374] EAE was induced in female C57BL/6 mice as described in
Example 1. Results are shown in FIG. 31A-B. On the day of disease
onset (score>1, dotted line), mice were treated with vehicle
only (daily, n=9)) or nalfurafine (0.01 mg/kg by i.p. injection
daily; n=8)(A). In a separate similar experiment, mice were treated
with vehicle only (n=5) or clemastine fumarate (10 mg/kg by i.p.
injection; n=7) (B). Mice were considered recovered if they
received a score<0.5 by day 23 post treatment initiation. Shown
are the percentage of mice in each group that recovered (A) and the
number of days in recovery (B). ****p<0.0001 by Fisher's exact
test (% recovered; A) or one-way ANOVA with Holm-Sidak's multiple
comparison test (# days in recovery; B). These data are from the
same experiments as Example 30.
Interpretation and Impact:
[0375] Clemastine fumarate, an anti-histamine which also
antagonizes the muscarinic receptor, has been shown to reduce
chronic disability in the EAE model when used at 10 mg/kg starting
at the time of immunization. Additionally, it has been shown to
enhance remyelination in mice and humans. In our EAE model,
clemastine fumarate treatment promotes recovery in just over 50% of
the mice but the recovery is not sustained. In contrast, all of the
mice recover when treated with nalfurafine and have a sustained
recovery. This finding indicates that nalfurafine is superior to
clemastine fumarate in this model and provides a more sustained
improvement in every animal treated.
Example 32: Nalfurafine Promotes Recovery in Pain Threshold when
Administered after Demyelination in the Cuprizone Demyelination
Disease Model of MS
Experimental Detail:
[0376] A demyelinating disease state was induced in female C57BL/6
mice (8-14 weeks older and between 17-23 grams in weight). The mice
were fed cuprizone-containing chow (0.3% (w/w) cuprizone) or chow
only (normal controls) for 35 days, at which point they were
switched back to standard chow. At day 28, mice were started on
daily treatment with vehicle only (DMSO: Tween 80: Saline) or
nalfurafine at 0.1 mg/kg by i.p. injection. In a second experiment,
mice were fed cuprizone-containing chow (0.3% (w/w) cuprizone) or
chow only (normal controls) for 42 days, at which point they were
switched back to standard chow. At day 35, mice were started on
daily treatment with vehicle only (DMSO: Tween 80: Saline) or
nalfurafine at 0.1 mg/kg by i.p. injection. In both studies, on day
70, mice were culled. See FIG. 32 A for an outline of the disease
induction and treatment time course.
[0377] Sensitivity to mechanical force elicits paw withdrawal in
mice. Threshold to withdrawal is measured using calibrated von Frey
filaments using the up-down method (Bonin et al. A simplified
up-down method (SUDO) for measuring mechanical nociception in
rodents using von Frey filaments. Molecular Pain. 2014; 10:1-11) at
maximum disease, prior to treatment with nalfurafine. Cuprizone
causes increased mechanical sensitivity compared to mice on a
normal diet ({circumflex over ( )}p<0.05) (FIG. 32B), and this
increase in mechanical withdrawal threshold is reduced to baseline
levels following treatment with nalfurafine (0.1 mg/kg/i.p.).
*p<0.05 at maximum disease (day 28 or 35) and following daily
treatment with nalfurafine (average threshold days 45-70).
Nalfurafine treated mice improved mechanical threshold scores
compared to vehicle treated mice(#p<0.05). Student t-test,
n=10-11 mice/group from 2 independent experiments. A compared to
mice on normal diet; * threshold pre and post treatment; #
differences in recovery between treatment groups. Pooled data from
2 experimental cohorts were analysed (max disease is week prior to
treatment initiation and at maximal recovery (days 63-70).
Interpretation and Impact:
[0378] Chronic pain is often associated with multiple sclerosis.
Allodynia is an increase in pain sensation to a normally
non-painful stimulus. In this test von Frey filaments are used to
measure the paw withdrawal threshold following application of a
defined mechanical force. Following cuprizone induced
demyelination, the pain threshold is a functional biomarker for
recovery, indicative of remyelination of the nerve fibres.
Remarkably, the diseased animals treated with nalfurafine showed a
pain sensitivity that was similar to baseline, indicating that
treatment enhances functional recovery.
* * * * *